At night, if you go to the Sanctum Sanctorum the back door will be open with purple fog spilling out.
When Spider-Man is hitting the Green Goblin,he says,"Why won't you fall down?". That is a reference to the comics when Spider-Man is fighting Ox and says,"Why won't you fall down?"
Early in development, plans were to have a Hulk and Spider-Man team up. However, the developers opted for Venom instead, because they felt he was more closely related to Spider-Man anyway.
In the first race against Human Torch, Torch says "Darkhawk is faster that you!" Darkhawk is an underrated b-list hero in Marvel comics
Due to the success of the game, writer of the game and comic of the same name, Brian Michael Bendis, made the game canon to the Ultimate Spider-Man comics, and artist Mark Bagley even added panels that were identical to the game.
The Nintendo DS version of "Ultimate Spider-Man" had a follow-up sequel to this game known as Spider-Man: Battle for New York (2006) where you play as both Spider-Man and the Green Goblin.
Many of the ratings that appear while performing combos (such as "Amazing", "Spectacular", and "Ultimate") are references to the titles of various Spider-Man comic book series.
When you are playing as Venom fighting Silver Sable's mercenaries, you come across a building with a big hole under reparations. This is one of the buildings Rhino ran through with Spider-Man chasing after him on a previous mission.
The little boy who loses his balloon is the first person that is devoured when using Venom. This is a nod to the Spider-Man 2 (2004) video game, where the task of returning the balloon back to the boy was a recurring goal in that game and was considered extremely annoying by game players who played Spider-Man 2.