Ledecky in charge but Slovak swimmer collapses
Canadian star Summer McIntosh was also back in La Defense Arena in the 200m medley as she seeks to further burnish her reputation, while Caeleb Dressel kickstarted his 100m butterfly defence.
American great Ledecky, who emphatically defended her 1500m title on Wednesday, came home in 8min 16.62sec.
Her chief rival Titmus, who came second in Tokyo, was third (8:19.87) in the same heat with the United States' Paige Madden second quickest.
Ledecky has already rewritten the history books in Paris with her 4x200m freestyle relay silver on Thursday making her the most decorated female Olympian swimmer with 13 medals, including eight gold.
It propelled her clear of fellow Americans Jenny Thompson, Dara Torres and Natalie Coughlin and Australia's Emma McKeon.
By winning the 1500m, she became the first woman swimmer to win gold at four Olympics.
And should she clinch the 800m, she will equal the most Olympic golds -- nine -- ever won by a woman athlete, matching former Soviet-era artistic gymnast Larisa Latynina.
"I mean, I've tried not to really think about it," Ledecky said of the records.
"I'm just taking it one event at a time, and I know I have challenges in each of my events. So when all is said and done, I'll enjoy it.
"And I've certainly appreciated all the support and cheers that I've felt as I've gone through the week."
She has the 16 fastest 800m times ever recorded, but a chink appeared in her armour this year when she was beaten for the first time since 2010, by McIntosh.
But the Canadian is not contesting the event in Paris, leaving Titmus as her main roadblock.
Conscious
McIntosh, a decade younger than Ledecky, already has two golds and a silver and is gunning for more glory in the 200m medley, where she topped the timesheets in 2:09.90.
Slovakia's Tamara Potocka came last behind McIntosh and collapsed as she climbed out of the pool.
The 21-year-old, at her first Olympics, received medical attention and was carried out on a stretcher with an oxygen mask on.
"She's conscious but we are waiting for a medical update," organisers said.
Australia's Kaylee McKeown, who swam the fourth fastest time in history in June, cruised through her medley heat in 2:11.26 ahead of defending her 200m backstroke title on Friday evening.
Defending champion Dressel, who shattered the world record in Tokyo, was sixth fastest through the 100m butterfly heats in 50.83sec.
But it was the man who finished second behind him three years ago, Hungarian Kristof Milak, who set the pace, hitting the wall in 50.19sec.
Dressel and Milak are the only active swimmers to ever dip below 50 seconds.
The American, who won five gold in Japan and has one in the relay so far in Paris, faces a big day with the semis in the evening, after he dives in defend his 50m freestyle title.
"Feeling good, yep, here to race," he said.
In other action, the United States qualified first for the final of the mixed 4x100m medley relay ahead of Australia and China.
Along with the 50m fly and 200 backstroke finals, French superstar Leon Marchand is in action again later, with expectations high that he can win a fourth gold of the Games in the 200m medley.
The 22-year-old has already won the 400m medley, 200m butterfly and 200m breaststroke titles.