The Much Ado About Nothing filmmaker told Vulture that he is too busy with upcoming projects - including the planned Avengers sequel - to write a follow-up to his 2008 web-series.
Asked if a sequel would emerge, Whedon said: "Not soon. [Much Ado] was already written. See, that's the thing. Directing is a joy, but it's the writing that really requires the energy."
Dr. Horrible's Sing-Along Blog starred Neil Patrick Harris as a kind-hearted super-villain and Nathan Fillion as his nemesis Captain Hammer.
Following its online release, the series was aired on television by The CW in October 2012.
Fillion previously told Digital Spy he was convinced that a sequel would eventually materialise, while The Big Bang Theory's Simon Helberg - who play sidekick Moist - also told DS that "everyone [involved] is excited" to start work on a follow-up.
> Joss Whedon on Dr Horrible sequel: 'We want to shoot it this year'
Watch a clip from Dr. Horrible's Sing-Along Blog below: