Park, who has dominated the event, touched first but was disqualified for a false start in a stunning blow to his hopes of defending the title he won at the 2008 Beijing Olympics.
Chinese rival Sun Yang capitalised on Park's misfortune by topping the qualifiers into the final later Saturday with a best time of three minutes 45.07 seconds.
Park had come through to overhaul British pacemaker David Carry and win his heat in 3:46.68, but the crowd gasped when the semaphore board flashed that the Korean had been disqualified.
"I don't know what happened," a stunned Park told reporters shortly after leaving the pool. "I need to speak to my coach to find out."
Park, a two-time 400m world champion, who spent time training in Australia as preparation for the London Olympics, has never been beaten by Sun in international competition.
Sun, the 1500m freestyle world record holder, now gets the chance to go after the 400m freestyle Olympic gold medal ahead of Americans Peter Vanderkaay (3:45.80) and Conor Dwyer (3:46.24).
While Park was coming to terms with his dramatic early exit there was also disappointment for German world record holder Paul Biedermann, who also missed out on the final with his 12th-fastest time of 3:48.50.
"It wasn't so good," Biedermann said. "I wanted to lead from the front, but I couldn't hold it.
"I have the 200m freestyle tomorrow and I am hoping for a better outcome."
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