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E.V.O. is a side-scrolling platformer action game where players must navigate a creature of their own design across a number of environments resembling Earth's past. The game is divided into five distinct geological periods: the Cambrian Period and the Ordovician Period of the Paleozoic Era ("The World Before Land") from 500 million years ago to 450 million years ago, then the Carboniferous period ("Early Creatures of Land"), the Mesozoic Era ("Age of Dinosaurs"), the late Neogene period ("Ice Age"), and finally the early Quaternary period ("Early Man"). Each period has its own map screen, which is divided into various levels. While each era takes historical liberties with both its inhabitants and time frames, the player's choices for evolution are dependent on the current era of play, ranging from aquatic bodies during the Age of Fish to mammalian physiology during the Age of Man. During the first two eras you are forced to gain a new body to progress, but once you reach the Age of Dinosaurs, a side quest will give you the option to remain as a reptile, or become a bird, gaining the ability of flight. As you enter the Ice Age as a bird or reptile, you are immediately given the option to become a mammal. You can finish the game as a reptile, bird, or mammal and the outcome is still the same, only the interaction dialogue is affected by your choices.

As the player progresses through each level, other organisms are encountered who must be confronted by biting, ramming them with horns, or jumping on them until they are defeated. Defeated enemies leave behind meat which the player can consume to grant them "evolution points" used towards upgrading specific body parts. The body of the character controlled by the player is divided into eight sections which can be upgraded by spending evolution points, making them stronger as well as changing their appearance. New abilities such as tail swings, greater jumping ability and increased movement speed can also be obtained through evolution.

During gameplay, the player will also encounter crystals that either display hints and tips, grant large amounts of Evolution Points or transform their character into a unique body for a limited time.As the player takes damage from enemy creatures, one may restore health at any time by either consuming the meat of their foes, eating nearby plant life, or by undergoing selective evolution. At the end of each level, the player must face a stronger boss character in order to progress, requiring much more effort to defeat than a typical foe. There is no game over mechanism, if the player's character loses all their health points the player is revived by Gaia and sent back to the game's map screen with a deduction of roughly half of their Evolution Points. The game utilizes a built-in battery backup save system for storing game data. The player may record progress directly to the cartridge to be continued at another time, and store up to fifty previously created creatures using the game's built-in "Book of Life" feature. Creatures stored in this index have the option to be recalled at any time when the player encounters special crystals during the quest.

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