Their team of Murphy, Nic Fink, Gretchen Walsh and Torri Huske touched in 3min 37.43sec to narrowly better the mark set by Britain at the Tokyo Games, when the event was first added to the Olympic programme.
China, led by Zhang Yufei, took silver in 3:37.55 with Australia filling the podium in 3:38.76.
It is only the second world record in the pool in Paris after China's Pan Zhanle smashed the men's 100m freestyle best.
"We didn't talk about the world record," said Murphy, who gave the United States a strong start in the opening backstroke leg.
"I think we know the potential of everyone on this relay and luckily tonight everyone swam to their potential and we got the world record and got the win."
Teams in the mixed medley comprise two women and two men, with each of the four swimmers allocated to one of the four traditional medley strokes -- backstroke, breaststroke, butterfly and freestyle.
The United States chose to lead off with Murphy up against China's Xu Jiayu with Australia opting for a woman in 100m and 200m Olympic champion Kaylee McKeown.
Both Fink and Walsh then swam storming legs before Huske brought it home with China's Yang Junxuan and Australia's Mollie O'Callaghan in hot pursuit.
"This meet has been great and this meant so much to me, the world record with these three is just like unbelievable," said Huske, the 100m butterfly gold medallist.
"I feel like they make it so easy to be confident because they're the best in the world. So I'm just so lucky that I get to have them by my side."
