About 53 other people were injured in the shooting, which took place in the Pulse Club on Sunday morning.
The gunman took hostages at the club and barricaded himself in the complex but was later killed by police.
"It appears he was organized and well-prepared," Orlando Police Chief John Mina said, adding that the assailant had an assault-type weapon and a handgun.
Authorities described the attack as a domestic act of terrorism. The Federal Bureau of Investigation (FBI) is also involved in the case.
A paramedic said the casualties will rise as more people are being brought out of the club.
A man who had been inside the club earlier described a situation of chaos outside as the number of casualties became apparent.
“There were just bodies everywhere,” he said. “In the parking lot, they were tagging them - red, yellow - so they knew who to help first and who not help first… Just blood everywhere.”
More than 100 people were said to have been in the nightclub when the shooter opened fire at around 2:00 am local time (0600 GMT).
"We heard rapid fire go off. In the room I was in, people went down to the floor. I wasn't able to see the shooter or people get hurt,” one witness said.
Video footage showed a high police presence around the club, accompanied by bomb sniffing dogs. Residents were warned by police to steer clear of the area.
According to the Mass Shooting Tracker, a total of 372 mass shootings - defined as a single incident that kills or injures four or more people – occurred in the US last year, killing 475 people and wounding 1,870 more.
The Sunday shooting came just only two days after a gunman fatally shot singer Christina Grimmie during a live event at the Plaza Live Theater in Orlando, before ending his own life.
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