The Japanese manual for Sonic & Knuckles alludes to the game's Special Stages in its story section, which includes a moment where Sonic gets sucked through a Super Ring and arrives at the Master Emerald Alter, where the player accesses the Special Stages in the game. However, writer Ian Flynn would later claim the Special Stages seen throughout the series are not considered canon, instead only existing as a gameplay mechanic.

Connecting Sonic & Knuckles to the first Sonic the Hedgehog will not allow you to play as Knuckles in the latter game. This was not due to hardware limitations as is commonly believed, but rather because the game's development team felt that, while Sonic 2 was designed with Super Sonic's flight in mind and thus had verticality to its levels fit for Knuckles' climbing, Sonic 1 was not, and therefore simply was not fun to play as Knuckles in.

Thrash the Tasmanian Devil, a character introduced in Archie's Sonic the Hedgehog comics, is based on a purple sprite that is created for several milliseconds on the game's title screen if the player quickly switches between Sonic and Knuckles' sprites. Notably, Ian Flynn stated in the graphic novel "Sonic Universe 3: Knuckles Returns" that Thrash was meant to be another rogue of Knuckles who could "throw down with Knuckles and have it mean something", which did not line up with his role in his debut during "Sonic Universe" Issue #11. However, this would later be conveyed during the "Endangered Species" story arc of the main comic, where it's revealed that he has a strong hatred towards the Echidna species due to their actions against his ancestors. When asked about the possibility of bringing Thrash back in the comic's reboot, Ian Flynn said he would love to bring him back, but "it comes with all that baggage... We can't really bring him back without people going 'Oh boy! And here we go!'"

In the Flying Battery Zone Act 1, there's an object known as "The Flying Battery" which can only be seen by taking a certain path. It's simply an obstacle that can be pushed and only appears once in the game.

The game was meant to be longer, but due to time constraints, the high manufacturing costs of a 34 megabit cartridge with NVRAM, and Sega's commitment to a major McDonald's Happy Meal promotion featuring a sweepstakes to win a copy of the game, the decision was made to split the game into two games, Sonic the Hedgehog 3, and Sonic & Knuckles. Evidence of this split can be found in the level select menu (sound test), where levels from the second game (such as Flying Battery, Mushroom Vally, and Sandopolis) can be found.

When you arrive at the Sky Sanctuary Zone, Knuckles will jump over a big gap and create a bridge for you, allowing you to move on. He then sits down, exhausted, having you run after Dr. Robotnik without him. But if you instead turn to Knuckles and press down, having Sonic (or Tails) duck, Knuckles will see this as a bow, as if you're thanking him, and he will proceed to wave you off, as if he's saying "just go!".