Certainly not the hardest, or hard at all really, but the boss from Sonic 2, Chemical Plant Zone is really annoying
The first time you get there you WILL die because there are two platforms that fall away and reappear with instadeath below. Sprinting at full pelt in there, like you always would do in a sonic game, you will automatically fall off the platform.
Then, if you get hit by the blob of water it drops you will almost inevitably fly into the dead zone for an instadeath.
You can easily kill it in 5 seconds if you have someone playing as tails, but I'm sure many a child must have had oodles of lives stripped away, it's such an unnecessary annoying boss and really badly thought out.