Next to versions on consoles and Nintendo DS, Ultimate Spider-Man was also adapted for the Game Boy Advance. It follows the same basic story as the other versions (reducing it to its bare necessities and changing many details) and presents it as traditional 2D side-scroller.

The game is a follow-up to the Venom storyline from the Ultimate Spider-Man comic series. Three months after his apparent death, Venom is back and causing trouble. Spider-Man finds his home neighborhood of Queens besieged by a gang of thugs while a group of mercenaries have set their sights on capturing Venom. The game's storyline is illustrated by comic-style cutscenes, with many of the images re-used from the DS version.

The game consists of seven parts called "issues" of three chapters each. As in the other versions of the game, players will control both Spider-Man and Venom, both of which have different abilities. To take out the many enemies, both have various attack moves at their disposal, which can be strung along into combos. Both can also stick to walls and ceilings and pull themselves up (Spidey using his web, Venom using his tendrils). Spider-Man can additionally use his webbing in various other ways, like immobilizing enemies by throwing a web ball and swinging across obstacles. His web fluid however is limited and must be filled up by collecting appropriate power-ups. Power-ups are also needed to refill his health. Venom on the other hand cannot find power-ups even though his health constantly drains. He instead has to feed on defeated enemies to restore his health. Some levels feature permanent power-ups: increasing Spidey's maximum amount of web fluid or unlocking new combat moves.

The main goal in each level is simply to find the exit, but there are other objectives that have to be met, sometimes with a time limit. Spider-Man in particular often has to rescue civilians or disarm bombs. A directional indicator displays where the next objective is to be found. Several of the levels consist purely of boss battles.