The Olympic bronze medallist, unbeaten this season, clocked the fastest time in the world this year of 52.83 seconds for victory.
Dalilah Muhammad of the United States was second in 54.09 and her compatriot and 2011 world champion Lashinda Demus took third in 54.27.
"To run under 53 seconds - it is fantastic. The reason for my improvement is that I changed coach and I now train with the boys," the 26-year-old told reporters.
"They are faster so they are my motivation."
After London 2012, Hejnova linked up with Dalibor Kupka, former coach to 2004 Olympic decathlon champion Roman Sebrle, and she counts European 400 metres gold medallist Pavel Maslak among her new training partners.
"It's a fantastic season for me. I'm still unbeatable... and I broke my personal best and the national record," Hejnova added.
It was only the second track gold for the Czech Republic at a world championships after Ludmila Formanova won the 800 in 1999.
American Demus went off fast and was the first to rise but Hejnova never let her rivals get too far in front, relying on her strength over the second part of the race to reel them in and leave them trailing.
Medal favourite Perri Shakes-Drayton was a disappointing seventh after suffering an injury to her left knee during the race.
"It was going all right until the first hurdle and after the first hurdle my knee just felt funny," the Briton said.
"It was going so well, leading the heats and the semi. But tonight after the first bend I was just going back and back and back."
Russia's Olympic champion Natalya Antyukh did not make the final.
(Writing by Alison Wildey, editing by Justin Palmer)
