Afghan-origin Omar Mateen, 29, of Fort Pierce, Florida, carried an assault rifle and a pistol into the packed Pulse club, which describes itself as "the hottest gay bar", about 2.00 a.m. on Sunday and started the deadliest mass shooting in the US history, CNN reported.
In addition to the people killed, he injured at least 53 others, police said.
"It appears he was organised and well-prepared," Orlando Police chief John Mina said. Authorities said they have not found any accomplices.
The IS has said it was behind the attack, but the extent of its involvement is not clear.
During the attack, Mateen called 911 to pledge allegiance to IS and mentioned the Boston Marathon bombers - Dzhokhar and Tamerlan Tsarnaev.
After a standoff of about three hours, while people trapped inside the club desperately called and messaged friends and relatives, police crashed into the building with an armoured vehicle and stun grenades. They killed Mateen, but by then he had killed 50 and left 53 injured.
Ten victims have so far been identified: Edward Sotomayor Jr, Stanley Almodovar III, Luis Omar Ocasio-Capo, Juan Ramon Guerrero, Eric Ivan Ortiz-Rivera, Peter O Gonzalez-Cruz, Luis S Vielma, Kimberly Morris, Eddie Jamoldroy Justice and Darryl Roman Burt II.
The attacker's former wife, Sitora Yusufiy, who lived with him for four months in 2009, said her family "rescued" her from the relationship when they became aware that he was being physically abusive.
She described Mateen, who worked for security company G4S, as "mentally unstable" and "disturbed".
The mass shooting has led to an outpouring of sympathy across the the globe, with people unfurling rainbow flags, holding vigils and lighting candles in tribute to the victims.
US President Barack Obama has described the attack as "an act of terror and an act of hate".
Obama has ordered flags on federal buildings to be flown at half mast until sunset on Thursday.
The president also postponed a joint appearance with the Democratic Party's presumptive presidential nominee, Hillary Clinton.
Pakistan's Prime Minister Nawaz Sharif said that shooting didn't "represent the will of a vast majority of Muslims."
Chinese President Xi Jinping, Indian Prime Minister Narendra Modi, British Prime Minister David Cameron, former Afghan President Hamid Karzai and Singapore President Tony Tan expressed their condolences.
In Seattle, a rainbow flag flew at half-mast on the Space Needle, while the One World Trade Center in New York was among a number of US buildings lit up in rainbow colours.
In France, members of the lesbian and gay community gathered at an art centre in downtown Paris on Sunday, placing burning candles in the shape of a heart and draping themselves in rainbow flags.
The attack took place during Pride month, said the ILGA, which represents 1,100 lesbian, gay, bisexual, trans and intersex (LGBTI) groups worldwide -- "a time of joy and celebration" for the gay and lesbian community.
According to the crowd-sourced Mass Shooting Tracker, the US last year suffered 372 mass shootings, defined as a single incident that kills or injures four or more people, BBC reported. Some 475 people were killed and 1,870 wounded.
The latest incident came as Orlando was still reeling from the fatal shooting on Friday night of 22-year-old singer Christina Grimmie following a concert in the city.