Like its predecessor, Donkey Kong Jr. is a platform game. There are a total of four stages, each with a unique theme. DK Jr. can run left and right, jump, and grab vines/chains/ropes to climb higher on the screen. He can slide down faster by holding only one vine, or climb faster by holding two. Enemies include "Snapjaws", which resemble bear traps with eyes; bird-like creatures called "Nitpickers", some of which can attack by dropping eggs; and "Sparks" which roam across the wiring in one of Mario's hideouts. DK Jr. can jump over these enemies while on platforms, switch from one vine/chain/rope to another to dodge them, or knock down pieces of fruit that will destroy every enemy they touch before falling off the bottom of the screen.
To pass the first three stages, DK Jr. must reach the key hanging next to his father's cage, whereupon Mario flees while pushing the cage off the screen. In the fourth stage, DK Jr. must push six keys into locks on the topmost platform to free Donkey Kong. After a brief cutscene, the player is taken back to the first stage at an increased difficulty. A bonus timer runs throughout each stage, and any points remaining on it are added to the player's score upon completion.
DK Jr. loses a life when he touches any enemy or projectile, falls too great a distance, touches the water and falls off the bottom of the screen or if the bonus timer counts down to zero. The game ends when the player loses all of their lives.