The left-hander became the first unseeded player to win the Wimbledon women's singles title in the professional era, leaving sixth seed Jabeur as the runner-up for the second successive year.
Jabeur was bidding to become the first Arab player to win a Grand Slam title and the first African woman to win one of the four majors.
"This is the most painful loss of my career," the crowd favourite said as she fought back tears.
"Today is going to be a tough day for me but I'm not going to give up and I am going to come back stronger.
"It's been a tough journey but I promise I will come back and one day win this tournament."
The unassuming Vondrousova had managed to win only one match in her previous four visits to Wimbledon and last year came with her wrist in a plaster cast to support a friend and go shopping.
But she beat four seeds during the fortnight, including number four Jessica Pegula in the quarter-finals and Ukraine's crowd darling Elina Svitolina in the semis.
"I don't know what's happening right now," Vondrousova, whose husband Stepan Simek was present for the final after spending the past two weeks looking after their pet cat Frankie back in Prague, said on court after receiving the Venus Rosewater Dish from Britain's Princess of Wales.
"This time last year I had a cast on so it's amazing that I can now stand here and hold this, it's crazy. I don't know how I've done it."
With the Centre Court roof shut because of the threat of rain showers, crowd-favourite Jabeur initially looked comfortable as she won the opening two games of the final.
But world number 42 Vondrousova, playing a canny game full of slow slices and clever angles, settled down and began to draw errors from her opponent.
Jabeur responded again to lead 4-2 but then seemed consumed by nerves as mistakes flowed from her racket and she dropped five successive games to lose the opening set
Vondrousova, sporting tattoos on her arms, led 1-0 and 40-0 in the second set and appeared to be marching to victory but Jabeur suddenly loosened her shoulders and found her range.
It looked as though Jabeur was getting on top as she won three games in a row, but Vondrousova never panicked.
Showing great court craft and subtle changes of pace she got back to 3-3 and as Jabeur's errors returned she broke serve at 4-4 to stand one game away from the title.
Reaching 40-0, she squandered her first match point with a double fault but put away a volley to claim the title at the second time of asking, appearing in disbelief.
Vondrousova, the first player to reach two Grand Slam finals as a non seed having contested the 2019 French Open decider, became the fourth Czech-born player to win the title in the professional era after Martina Navratilova, Jana Novotna and Petra Kvitova.
