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."

Number Seven Boat Trips and Bristol Ferry Boats both operate services in the city's harbour. Number Seven Boat Trips said the changes were "unjust".
Bristol Ferry Boats disagreed and said the new timetable was "fairer than the previous one".
The city council said it was introduced "following feedback from customers".
Both companies operate the same route between Temple Meads and Hotwells, at staggered times.
Previously, the two boats collected people every 10 minutes in a 20 minute slot - and then one boat picked up passengers over the next 40 minutes.
Changes now mean ferries collect at regular 20-minute intervals and passengers can now stay on the same boat for the whole trip, instead of changing in the city centre.
Richard Rankin, of Number Seven Boat Trips, said: "We're told to move from our timetable on the Hotwells route - which we've run for 15 years - 10 minutes earlier.
"What it does then is, by running two boats close together it maximises the potential for what passengers there are to get on the other side's boats.
"That strikes me as being very unjust."
But Philippa Bungard, from Bristol Ferry Boats, said feedback indicated the service was "better for everybody".
"[It] is much fairer than the previous timetable and everyone so far seems very happy," she added.
In a statement, the council said the changes were made to manage journeys in the "most effective way".
"The changes have been made following feedback from customers using the services, to ensure timings between the two do not overlap and deliver the best experience for visitors," they explained.
Videomakers Ethan and Hila Klein, who run the YouTube channel h3h3 Productions, have won a legal battle over copyright in online video.
[NEXT_CONCEPT]
A ferry boat firm in Bristol is claiming a new timetable "imposed" by the council favours its rival.