The Venture Bros. was originally conceived as a comic book story for an issue of Monkeysuit, but creator Jackson Publick realized his notes were too extensive for a short comic and proposed it as an animated series to Comedy Central, which rejected it.

The first draft of the pilot script was written in spring 2000, but the show was not greenlit until around summer 2002 by Adult Swim, and Publick had not originally considered Cartoon Network because he didn't want to tone the show down and was unaware Adult Swim existed.

The considerable delay between season one and season two was partly caused by Adult Swim taking time to decide on renewal, and partly because the show is drawn and inked in traditional animation style (digitally), making each episode time-consuming to produce.

The show's principal reference is Hanna-Barbera's Jonny Quest, with Dr. Venture imagined as what Jonny Quest might have grown up to be, Brock Samson modeled on Race Bannon, and the Venture boys based on the Hardy Boys standing in for Jonny and Hadji.

Because Warner Bros. Entertainment owns the rights to Jonny Quest through Hanna-Barbera, the show could not use the original characters directly, so Jonny Quest appears as 'Action Johnny' and Dr. Zin as 'Dr. Z'.

The mysterious Guild leader 'The Sovereign' is revealed in episode 26 to be real-life rock star David Bowie, though a later episode reveals The Sovereign is actually a shape-shifter who frequently takes Bowie's form.

Creators Jackson Publick and Doc Hammer have repeatedly described failure as a central theme of the series, calling it 'beautiful sublime failure' and stating that every character is flawed and 'sucks at what they do' yet is beautiful in that failure.

A commentary track recorded by Publick and Hammer for the season one episode 'Home Insecurity' was omitted from the Season One DVD due to space limitations and minor sound quality issues, though Publick said it could be downloaded separately, becoming known as the 'lost DVD commentary'.

The Season One DVD reached number 1 on Amazon's top-selling DVDs list on May 31, 2006, and its packaging and interior art were created by comic artist Bill Sienkiewicz.

A soundtrack album titled The Venture Bros.: The Music of JG Thirlwell was released alongside the Season 3 video release, featuring 20 tracks on CD and 16 on vinyl LP.