The game revolves around a Program designed and named by the player. The story begins with the Program battling Quorra with light cycles. The Program wins and goes to Tron City where he/she meets Quorra. The Program and Quorra become friends. Quorra finds Zuse who takes them to Tron's palace to find a trainer for the Program. They meet with Tron, and he fights the program to see if he/she is championship material. Afterwards, he tells the player's program that if he/she wins the championship he/she would be the first ISO champion. This could give ISOs someone to look up to.

Tron: Evolution serves as a precursor to the movie Tron: Legacy and sequel to both the graphic novel Tron: Betrayal and the Nintendo Wii and DS versions, Tron: Evolution - Battle Grids. It is designed to be an integral part of the Tron story, featuring certain characters and settings from both films. Evolution explains the events that led to Kevin Flynn's imprisonment inside the Grid,[1] as well as telling how the Grid evolved through the years. The player controls a Monitor program named Anon (short for anonymous), a security program written by Flynn to investigate a conspiracy in the world of Tron.