The game offers both single-player and two-player cooperative multiplayer modes as players progress through a series of levels. Featuring six playable characters—Ratchet, Clank, Jak, Daxter, Sly Cooper, and Bentley—the game comprises a total of 50 levels categorized into five "archetypes", five weapon classes, and four environments. These environments include Paris from Sly Cooper & Bentley, Metropolis City on Kerwan from Ratchet & Clank, Haven City from Jak and Daxter, and Planet X, an original game-created environment. Players have the flexibility to choose their character for each level, with each character offering a distinct gameplay style and potential advantages on specific levels. In cooperative play, the second player assumes the role of the first player's sidekick character. For instance, if the first player selects Jak, the second player automatically becomes Daxter. The five weapon classes—Disc, Whip, Bowling, Projectile, and Melee—each bring a unique gameplay element. The Disc is thrown akin to a frisbee, with players controlling its navigation remotely using the Move controller to achieve objectives like hitting moving targets. The Whip is operated via the Move controller with 1-to-1 movement. Bowling functions similarly to the Disc, where players throw a bowling ball and guide it using the Move controller. Projectiles involve various guns and weapons, such as grenade launchers. Melee utilizes each character's distinctive weapons—Sly's cane, Ratchet's wrench, or Jak's gunstaff—to engage enemies in a manner reminiscent of their original games.

After an evil force has stolen entire chunks of the hero's home worlds and enslaved a race of peaceful space creatures, the heroes find themselves locked in a struggle for their survival