The 19-year-old, who won gold medals in Berlin in the 100 breaststroke and the 4x100 mixed medley, clocked a time of 26.62 seconds.
The Englishman's effort eclipsed the previous record of 26.67 seconds set by South African Cameron van der Burgh in Rome in 2009.
"I feel absolutely amazing. I had to look at that scoreboard about five times before I knew I had broken it," Peaty said.
"I haven't broken a world record before so I don't know how it feels. Hopefully, it will sink in by Saturday so I can get my head down and hopefully get a better performance out of myself in the final.
"It was not a perfect race due to my final touch so I can still improve tomorrow."
Peaty also claimed two golds and a silver at the Commonwealth Games in Glasgow last month.
(Writing by Tony Jimenez, editing by Ed Osmond)
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