Mike Tyson's Punch-Out!! is the story of Little Mac, a 107 pound underdog from the Bronx who wants nothing more than to be the World Video Boxing Association's champion. The 17-year-old must fight his way through the ranks of three circuits before facing the man himself, Mike Tyson.

Along the way, he'll face ten different opponents in 13 matches. The opposition is a varied group of comical fighters from all over the world, ranging from Glass Joe, a 110 pound weakling from France, to Super Macho Man, the 242 pound W.V.B.A. champion. Little Mac is greatly outmatched, so he'll have to figure out the best strategies to counter the brute strength and tricky techniques of his opponents.

Each boxer has a energy meter that is decreased with every punch received. When a boxer's energy meter is emptied, they hit the canvas. Matches can be won by either a traditional or technical knockout. A TKO is achieved when a fighter is knocked down three times in one round. Each round lasts three minutes.

Little Mac can block, duck, and dodge left and right. He can punch to the face and execute body blows. Powerful uppercuts can also be performed if Little Mac has a star. Stars are earned by catching your opponent off-guard.

Hearts keep track of how many punches Little Mac can throw, and his number of hearts decreases when he is hit or has one of his punches blocked. When he runs out of hearts, he becomes weak and cannot throw a punch. Only by dodging can he survive. If he is knocked down, pressing 'A' rapidly will make him get up.

When Little Mac becomes the champion of each circuit, he works out on the streets of New York with his trainer Doc Louis and receives a password that allows the game to be resumed at a later date.