The pair were sued after making fun of a video by another film-maker, Matt Hosseinzadeh, in which he pursued a love interest using parkour.
They had used clips of his film, which he claimed was copyright infringement.
But a judge ruled the Kleins' video criticising his work was "fair use as a matter of law".
Ethan and Hila Klein have an audience of more than 4 million subscribers on their YouTube channel.
In February 2016, they posted a video in which they watched Mr Hosseinzadeh's parkour video and made fun of it.
Their video included several clips of his original video, interspersed with their own commentary and jokes.
Mr Hosseinzadeh - known online as Matt Hoss - said in his original complaint that the pair had reproduced "virtually all of the work" as "nothing more than a prop" in their comedy routine.
But on Wednesday, New York district judge Katherine Forrest rejected the claim.
"Any review of the Klein video leaves no doubt that it constitutes critical commentary of the Hoss video," she wrote.
"There is also no doubt that the Klein video is decidedly not a market substitute for the Hoss video."
The ruling is significant since so-called reaction videos, in which YouTubers use clips of others' work and react to them, are a popular genre on the platform.
Forrest warned that while the Kleins' video was fair use, other reaction videos were often "more akin to a group viewing session without commentary".
"Accordingly, the court is not ruling here that all 'reaction videos' constitute fair use," she said.
Mr Klein said the ruling was a "huge victory for fair use on YouTube".
He told the BBC: "All of our work for the past four years is validated, as there has never been a court opinion about reaction videos.
"The judge called our method of criticism 'quintessential', which is a huge win for us and the YouTube community. I'm extremely grateful to everyone who supported us along the way."
Videomakers Ethan and Hila Klein, who run the YouTube channel h3h3 Productions, have won a legal battle over copyright in online video.