That One Boss: The Beast at the end of World 2. It's a pain in the patookie for two main reasons: 1) The Beast is fast, agile, and adroit. The Boy is not. 2) No matter what he gets attacked with, the Boy dies in one hit, and if he does, then you have to start the whole fight over again from scratch. And you can't quit mid-fight and pick up there, either—if you want to stop, you have to go through the whole level over again.
It is possible to glitch the boss out and make the fight unwinnable by removing a spike ball. Normally they regenerate if they do not hit the boss, but when this troper played the level, he got all of the spike balls to disappear and the boss was still alive. He waited at each spawn point to confirm it.