According to a 2009 interview with series creator SUDA51, this game was originally meant to be the last to feature Travis Touchdown as the series protagonist. However, due to the delayed production of subsequent entries Travis Strikes Again: No More Heroes and No More Heroes III, he changed his mind, instead wishing to focus on how much older Travis had become since the second game.

Shinobu's outfit in No More Heroes 2 is based off the Japanese "Gothic Lolita" style.

Before facing Kimmy Howell, parts of the Charlie Macdonald's mecha, Santa Death Parade, can be spotted around the now-damaged Santa Destroy University.

Kimmy Howell's in-game skirt is longer than the one she wears in her PR Illustrations and concept art. In-game, her skirt is right above her knees, while the one she wears in the illustrations stops at the thigh area and resembles those of a miniskirt.

Margaret Moonlight's theme, "Philistine" plays normally in the American version of No More Heroes 2: Desperate Struggle. However, in the Japanese and European version of the game, a remixed version of the song plays during the boss battle.

Alice Twilight's weapon "ASURA-6" is named after the Asura, famous for having up to 6 arms.

In an interview with Nintendo Power, SUDA51 revealed that Nathan Copeland was originally meant to appear in the first No More Heroes, but dropped him in favor of Death Metal. SUDA commented that, "When I started working on this installment, I knew right away that I wanted to bring Nathan back."

Margret's last name does not appear (outside of the credits) in the American release of the game. Her listing is just simply titled "Margret" and when she dies the screen just simply states "Margret is Dead". This is quite strange as every other release of the game states her full name, Margret Moonlight, much like Alice Moonlight.
This was proven to be a dubbing error as Alice's real last name was Twilight and is corrected in all other releases.