The American teenager broke the Czech fourth seed at 5-5 in the final set of the third-round match.
Keys, coached by three-time Grand Slam singles champion Lindsay Davenport, screamed with delight on sealing victory in Melbourne.
The world number 35 plays Madison Brengle in the fourth round as four United States players made the last 16.
"My hands are still shaking, right now I can't even process this, I'm just so excited," said Keys.
Kvitova, winner of Wimbledon in 2011 and 2014, has never made it past the last four of the Australian Open.
Last year she was beaten in her first match by Thailand's Luksika Kumkhum, playing in just her second Grand Slam tournament.
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