Thorpe, who also won the 400 and 200 individual freestyle, took over just ahead of the Americans and pulled away from their final swimmer Klete Keller to bring Australia home in seven minutes 08.58 seconds.
Michael Phelps, who could rival Thorpe as the most prolific winner of the world championships, claimed his first gold medal in his bid for four individual titles when he retained the 200 metres butterfly crown in rousing style.
But the 18-year-old American had to settle for silver in the relay despite edging out Grant Hackett, bronze medallist in the individual 200 freestyle event in the first leg. The U.S. finished the best part of two seconds behind the Australians in 7:10.26.
James Gibson claimed Britain's first individual world championship gold medal for 28 years in the men's 50 breaststroke, while Alena Popchenko put Belarus on the winner's podium for the first time ever in the women's 200 freestyle.
Thorpe's tally of world golds comprises two in 1998, an unprecedented six in 2001 and three in the Palau San Jordi, with four more days of competition to come.
Michael Gross, West Germany's outstanding performer of the 1980s, is the only swimmer to have won more world medals than Thorpe, having collected 13, of which five were gold. Americans Matt Biondi and Jenny Thompson have 11, like Thorpe, but not all are gold.
Thorpe, who qualified for Thursday's 100 freestyle final with the third-fastest time of 48.71, a personal best, earlier on Wednesday.
"It's been quite a while since I did a PB, so I was really pleased
He also contests the 200 individual medley (IM) and 4x100 medley relay in Barcelona, while Phelps clashes with the 20-year-old Australian in the 200 IM and swims the 100 butterfly, 400 IM and medley relay.
Phelps, who broke his own world record with a time of 1:53.93 in the 200 butterfly semi-finals, could not quite match that on Wednesday, though nobody could match him.
Phelps won by over a second from fast-finishing Takashi Yamamoto of Japan, clocking 1:54.35, the second-fastest time in history and inside the 1:54.58 old world mark.
Yamamoto, fourth at the final turn, charged through to overtake Olympic champion Tom Malchow and snatch the silver in 1:55.52. Malchow, silver medallist behind Phelps in 2001, was squeezed out to the bronze in 1:55.66.
Commonwealth champion Gibson, bronze medallist in Monday's 100 metres breaststroke, pulled ahead in the 50m near the end of a close race to win in 27.56 seconds from defending champion Oleg Lisogor of Ukraine. He became Britain's first individual world champion since David Wilkie in 1975.
"I've been working for this for four years," Gibson said. "I felt very relaxed, very chilled out."
Lisogor clocked 27.74, way outside his 27.18 world record, for the silver and Hungary's Mihaly Flaskay took bronze in 27.79.
In the women's 200 freestyle, experienced Slovak Martina Moravcova set the pace over the first 150 metres, but Popchenko, the European bronze medallist, accelerated out of the final turn to win in 1:58.32.
Olympic silver medallist Moravcova had to settle for another silver in 1:58.44 and China's Yang Yu took bronze in 1:58.54.