Hazzard County is having a reunion festival, which includes a Toughman-style contest, and a rattlesnake chili cook-off, along with several other events. Uncle Jesse is the organizer of the reunion and at the same time, is facing a lawsuit filed by Mama Josephine Maxx for access across Duke property to the Hazzard swamp where she intends to build a theme park. Jesse however, refused access, not wanting to see the swamp destroyed. This however, has made the rest of the townsfolk angry with him, as the new theme park means new jobs, and revenue for Hazzard County.
Driving from Washington, D.C., the Congressman representing Hazzard County in Washington, Cooter Davenport, arrives first and goes back to the Duke farm with Jesse, where Jesse is barbecuing ribs for everyone. Shortly thereafter, a car pulls up. Jesse recognizes it as Bo due to the sound of the motor. Bo has gotten a ride from a friend as he, having returned to the professional racing circuit and has come back for the reunion, has wrecked his car (again, according to Jesse). Bo introduces the girl, named Tammy, who brought him home and after she leaves, Cooter says she was so good looking, it hurt and Bo joked with him, saying the girl wasn't bad either. Then Daisy arrives on a motorcycle. Daisy has been at graduate school getting a PhD in ecology after a bitter divorce. Then shortly after Luke, who has become a smoke jumper for the forestry service, arrives on a fire truck. He stops to kiss a fully-suited firefighter, whom Cooter at first thinks is another guy, and wondering out loud how long Luke has been away from Hazzard. Then the other person's hat falls off, revealing a blonde-haired woman underneath the uniform and the Dukes get a laugh at Cooter's expense.
Now that everyone has arrived, Jesse tells the story of Mama Maxx's lawsuit and everyone is shocked for a moment to hear that Rosco is now the county Boss as well as Sheriff. Jesse tells them that Rosco inherited J.D. 'Boss' Hogg's "empire" when he died a few years ago.
When the Dukes arrive at Hazzard, they soon thereafter get suckered into an overland car race to determine whether or not Mama Maxx gets access to the swamp across Duke property. So Bo, Luke and Cooter go to get the General Lee, which has been stored for years in Cooter's abandoned garage. Problem is, when they arrive there is no car there. A moment later, Enos arrives from Los Angeles, where he has returned to work in the LAPD. When Bo and Luke suggest the General has been stolen by Mama Maxx, Enos notes that there are no tire tracks on the floor so the General must have disappeared a long time ago. He then leaves to go to the Duke farm to see Daisy.
Cooter takes Bo and Luke to the town garage which was run by a man named Red, whom Cooter gave a bunch of junk cars too that was in the same garage. Cooter figures Red took the General Lee by mistake. When they arrive in town, they find that Red has retired and left his daughter Mavis in charge of the garage. She says that her dad sent all those cars, except the General cause her dad thought it too pretty to destroy, to the car crusher. They find the General in the back of the garage covered with chickens, roosters and feathers and such. After an all night session, the General is restored and ready to go. Bo and Luke take the General for a test run, find they can still outrun Rosco and Cletus, only to have the car overturned by an unknown assailant.
That night, Cooter drops the Dukes off at the relocated Boar's Nest (as the original one was destroyed in a wedding reception) and he takes the General in to start working on it again. Bo and Luke go in and sit with Enos and Daisy, who are on a date that night, and Bo recognizes the men who ran them off the road, including a disbarred race driver named Kam Cutler. They realize that Mama Maxx has hired Kam to help them win the race. After a bar fight with Mama Maxx's men, in which the Dukes (and Enos throwing Riker, one of Mama Maxx's men, out a window) win rather convincingly, they return to Cooter's where he warns them that another stunt like what happened and the General Lee will be nothing but an orange pile of parts.
When Luke begins considering a few dirty tricks of their own, he's stopped by Jesse, who says that they'll run the race fair and square and that no causeway can be built to the Hazzard Swamp because the ground will not support such a thing, due to the lack of supporting bedrock. Jesse points to a survey that the Army Corps. of Engineers made when they planned the same thing in 1941 as a shortcut to Fort Hooker, only to abandon the idea, he also mentions that Mama Maxx knows about it.
At the Hazzard Swamp, Daisy and Enos have a picnic and Enos confesses the only reason he returned to Hazzard was that, after a tour of duty on the Drug Task Force and the SWAT team, he has finally gotten the courage and he asks Daisy to marry him. She then finds an unknown type of fern, and then, almost as an afterthought, accepts Enos' proposal.
Later that day, the Dukes take a Federal Ecological report to Rosco and tell him if he builds on the swamp without filling the government form they've brought to him out, he may face a federal investigation. Then Daisy and Enos ask if Rosco will hurry their marriage license through quickly and ask him to perform the ceremony, which he agrees to do. Left alone, Rosco laments that his mother said that Enos would get married before he did.
At the same time, Mama Maxx's men are telling her that they saw Daisy find some gallium arsenide ore in the swamp, their having been there the same time Daisy and Enos were and observng the lovebirds from a distance, and having mistaken Daisy's examination of the fern as her anilyzing a soil sample. So she orders Daisy to be kidnapped. As soon as Daisy leaves a shop in Hazzard, she's grabbed from behind, gagged with a handkerchief and thrown in a van, struggling and squealing all the way. Jesse receives a phone call saying that she will be released after the race, but only if Bo and Luke lose.
When Enos arrives at the Duke farm, Luke and Bo tell him about Daisy's kidnapping and that they need to make a plan to get her back. Meanwhile, Daisy has spoken to Mama Maxx and managed to manipulate her into letting her men continue to run errands for Daisy's wedding because if plans suddenly stop then everyone will know something is wrong. She reluctantly agrees and then after a passionate request from Daisy, agrees to help with the wedding plans herself. Daisy eventually asks Mama Maxx to be her matron of honor.
On the day of the race, Jesse's rattlesnake chili takes second place, both Bubba and Bertha Jo reach the finals of the "off the street" boxing contest which causes Bubba to throw the fight and causing a riot at the contest. Meanwhile, the race begins between Kam in Buzz's "Double Zero," the only car to beat the General Lee in an overland car race, and the Dukes. The "Double Zero" gets far enough away from the General Lee to pass through the first checkpoint and then Kam switches with Buzz and takes off in a car made up like the "Double Zero" with an illegal stock car engine. With this engine, Kam sticks to the roads and takes it easy, figuring the race is already won.
The Dukes, after barely evading a trap set by Mama Maxx's men, head off the road and over to the cotton mill, where they have found Daisy at. When Luke sneaks in to see what they are up against, he sees that she's hired mercenaries loaded with arms and that Bo and Enos needs to go get help. Further down the road, Bubba and Bertha Jo are about to fight for real, but it is interrupted by Bo's timely arrival and he recruits them to help out. Fortunately for them, Mama Maxx has had a soft moment and has made the mercenaries give their guns up and only fight hand-to-hand or with whatever they can pick up. Ryker tells the mercenaries, "Hey, they're just hicks." Almost as soon as they are finished handing over their weapons, Enos, (armed with a sledgehammer and his bullet-proof vest from the SWAT team), Bo, Bubba and Bertha Jo all bust in through the door. They manage to overpower most of the men and then Luke dives down from the ceiling and helps finish them off. Enos rescues Daisy, Luke picks up Ryker and puts him in the General's trunk and then they take off to finish the race.
Heading along, with almost no hope of winning, the Dukes and Enos spot two "Double Zero's" on the side of the road. They pull over to see what is happening. Buzz comes out and tells Bo and Luke they have been racing Kam's car with an illegal engine and that he was supposed to finish the race in Buzz's car, but now it will not start. Especially without the distributor rotor that Buzz took off the engine to keep it from starting. When Kam begins to rush Buzz, Luke tells him he might want to leave Hazzard while he still can. With that, Kam jumps in his car and takes off out of the county. Buzz says he just wants to race the General Lee once more and fair and square to the finish line. Bo and Luke agree and they all get ready to take off. Luke figures that if they win, the people in Hazzard are going to be plenty upset, but if Daisy can get dressed for the wedding, they might not lynch everyone. When Buzz gets in position, at the sound of the General's horn, they take off.
Buzz decides to stay on the road and go straight in, forcing the Dukes to take the General overland. Bo cuts the General Lee through every shortcut and field he can find and think of, making three jumps in the process before getting close to town, throwing Ryker around in the trunk in the process. Once in town, it looks like Buzz is going to win, but the General races in from the other side of town and slides in front just in time to win the race.
While Cooter and Jesse celebrate, the rest of the disappointed townsfolk start to walk away until the Dukes pull up in front of a shocked Mama Maxx and Rosco. Luke pulls Ryker out of the trunk and tells Rosco he kidnapped Daisy on Mama Maxx's orders so they would lose the race. An enraged Rosco tells Mama Maxx that she does not have the brains of a turkey and he is sorry he conned her into building that theme park. She does not believe it, so he explains:
Rosco obtained a sample of gallium arsenide ore, ostensibly to be used in a mineral exhibit at the Hazzard High School, and put it in the swamp so Mama Maxx would find it. He also reveals that he knew she was a con artist as the FBI had sent him a list of the ten most wanted real estate con artists in the country and Mama Maxx is #1. When Bo and Luke asked who helped Rosco come up with this plan, he reveals that Boss Hogg helped him. When everyone in town is shocked by that revelation, Rosco explains that before he died, Boss wrote his memoirs called "The Best Laid Plans" and in that manuscript the late Boss Hogg wrote: "When picking a sucker, always pick a con artist 'cause they're the easiest to fool 'cause they're always trying to get something for nothing."
Before Rosco can arrest Mama Maxx, Daisy stops him, telling Rosco that he cannot arrest the matron of honor before the wedding. Mama Maxx tells Daisy that if she had not found that gallium arsenide ore, she would not have ever kidnapped her. Daisy then says she found a new fern species. Before they can go into detailed explanations, Jesse yells out, "Let's have a wedding!" and they all go to the town square. Daisy stands with Bertha Jo as her maid of honor and Mama Maxx with her too and Enos has Bo and Luke as best men. A nervous Rosco starts to recite a funeral speech before he is interrupted and changes pages and almost swears Enos and Daisy in as Deputy Sheriffs before he has stopped again. Before he can start a third time, a bus pulls up and the driver steps out. It turns out to be L.D., Daisy's ex-husband who is trying to get directions and when he realizes he is in Hazzard, he really knows he is lost. Daisy confronts him, asking what he is doing and he tells her he is driving a bus for a little country band. Daisy then passes out and when she's revived, she realizes that in seeing L.D., she is not yet ready to get married yet. Enos seems to understand, saying that when she threw an apple peeling over her shoulder earlier, it formed an "S" for Enos Strate and that they are destined to be together. He then says he will just have to wait for Daisy to grow up.
Daisy then asks if anyone wants a free wedding and Bo and Luke suggest Bubba and Bertha Jo get married as they already fight good enough to be married. Daisy then throws the bridal bouquet and Bubba catches it. Bo tells Rosco not to throw away that book just yet. And as the townsfolk heads back toward the square to see Bubba and Bertha Jo get married, Uncle Jesse turns to the screen and (in an occurrence of breaking the fourth wall), tells the audience: "Y'all come back now, you hear?"
The movie ends with the message "In loving memory" containing a list of actors and cast members who had died before the movie was produced followed by the Dukes and everyone else bowing play-style and stepping backwards one step before the end credits appear.