After dumping Batman underwater, Joker turns to Captain Clown and says, "Well, Captain, this could be the start of a beautiful friendship!"—a reference to the classic film Casablanca.
The computer readout describing the gas misspells "airborne" as "air born".
The newspaper delivery van at the very top of the episode bears a Gotham Globe logo, but the newspaper it delivers is the Gotham News.
When the Joker slides down the garbage chute, he first jumps down feet-first, but he comes down head-first at the next scene.
After Batman rushes upstairs and sees Alfred exposed to the gas, he apparently breathes it, coughs, and quickly puts his gas mask on. However, it is shown earlier that the gas has an immediate effect once it's inhaled, so the same thing should have happened to Batman.
"The Last Laugh" has been used as several terms on the video game DC Universe Online, including: a mission, a story arc and downloadable content.