Titmus' 200m final was billed as another match race against US great Katie Ledecky.
Ledecky finished fifth, with Titmus' main challenge coming from China's Hong Kong based swimmer Siobhan Bernadette Haughey.
Haughey held the lead until the last 20 metres.
But Titmus finished in a flurry to achieve a nail-biting win in an Olympic record time of one minute and 53.50 seconds.
Haughey (1:53.92) took silver with Canadian Penny Oleksiak (1:54.70) claiming bronze.
Titmus joins Shane Gould in 1972 as Australian woman to complete the 200m-400m golden double at an Olympics.
She will meet Ledecky once more at the Tokyo Games, over 800m, with the American the red-hot favourite over the longer distance.
Shortly before Titmus’ win on Wednesday, Australia also won two gold medals in rowing.
With the event returning to the Olympic rowing schedule after almost a 30-year absence, the Australian women's coxless four crew of Rosie Popa, Lucy Stephan, Annabelle McIntyre and Jessica Morrison stormed to gold.
The Australians held off the fast-finishing Netherlands crew, while Ireland won bronze.
The Australian men's coxless four crew of Alex Purnell, Spencer Turrin, Jack Hargreaves and Alex Hill made it a golden rowing double with a gold in their event.
While Australia won gold in the men's four in 1992 and 1996 to earn the ‘Oarsome Foursome’ tag, Great Britain had won the event at every Olympics since.
The Aussies crossed the line in an Olympic best five minutes 42.76 seconds, with Romania 0.37 seconds behind.