China took the bronze, with Canada fourth.
Ledecky now has 13 Olympic medals - eight golds, four silvers and a bronze - one more than compatriots Jenny Thompson, Dara Torres and Natalie Coughlin and Australia's Emma McKeon.
She and her great compatriot Michael Phelps are the only swimmers to win 13 medals, with the latter's tally of 28 in no danger of being matched.
"It's just amazing to get to be a part of even one-13th of the journey that she's been on," Ledecky's teammate Erin Gemmell told reporters.
"I think it's so much more fun to be on a relay than to be by yourself. We just had a great time out there tonight."
The Australian team of Mollie O'Callaghan, Lani Pallister, Brianna Throssell and Ariarne Titmus finished in an Olympic record time of seven minutes 38.08 seconds at the La Defense Arena.
Jamie Perkins and Shayna Jack, who swam in the heats, also took gold.
The U.S. finished 2.78 seconds behind with Ledecky putting in a fighting third leg to haul teammates Claire Weinstein, Paige Madden and Erin Gemmell up from third to second.
Another win would have taken Ledecky's tally to nine golds and level with former Soviet gymnast Larisa Latynina's record haul for a female Olympian from any sport, but that was too big an ask.
Ledecky can still do that in the women's 800 metres free on Saturday, after winning the 1,500. The American, who made her Olympic debut at London 2012, holds the world record in both distances.
Led from the start
On Thursday night Australia led from the start with O'Callaghan powering down the pool before handing over to Pallister, recovered from COVID earlier in the week and consolidating for Throssell to keep Ledecky safely at bay.
"This morning, we were wearing masks just to make sure. She (Pallister) has tested negative now, but I think to do those two incredible swims in one day is such a testament to her," Brianna Throssell said.
"I guess we just supported her as best as we could while also making sure that our health and safety was first as well."
Titmus was never in any danger as she made sure of her second gold and third medal of the Games.
"It was really fun. I feel like a bit of redemption for us. Tokyo was definitely not the result we wanted and I personally wasn't happy with how I performed in the relay in Tokyo," said Titmus.
Australia finished third in that race won by China, with the Americans second.
"I feel like I have a role to play in this team and do the best job I possibly can. And I think I did that tonight. I'm proud that they had faith in me... to put me last and get the job done," added Titmus.
