The defeat was a huge blow for the Serb, who will bid to join Roger Federer (103) and Jimmy Connors (109) as players who have won at least 100 titles when he heads to the Australian Open, where he will also seek a 25th Grand Slam trophy.
Djokovic saved three break points in stifling conditions to go 5-4 up but lost the opening set in a tough tiebreak as Opelka cranked up his serve and seized early control of his maiden tour meeting against Djokovic.
Opelka broke for a 3-1 lead in the next as Djokovic looked rattled by the 6ft 11 in (211cm) American, who pulled off some spectacular winners on both flanks.
The final blow came on Opelka's serve and he fittingly finished the match with a powerful ace.
Up next for Opelka is Giovanni Mpetshi Perricard in what promises to be an explosive battle after the Frenchman sent down 19 aces in his 7-5, 7-6(5) win over Czech Jakub Mensik to take his tournament tally to 75.
"He's the greatest player the sport has ever seen," said Opelka, shortly after one of his biggest wins.
"It's difficult being in Novak's position. He can scout me or other opponents all day long. The reality is we have nothing to lose against him.
"You end up playing more free. You take a lot more risk because it's your only chance. If you play your normal level or even above it he's going to win every time.
"He gets guys that are good players who roll the dice. And on a day like this, when a lot of things go my way, that's how it works out."
Defending champion Grigor Dimitrov went through after Jordan Thompson retired while trailing 6-1, 2-1, while Jiri Lehecka made quick work of Nicolas Jarry, securing a 6-4, 6-4 victory.