Flags on the Sydney Harbor Bridge flew at half-mast, and giant television screens were set up in Irwin's home state of Queensland for people to watch the service.
Up to 300 million people worldwide were expected to tune into the public memorial service.
Irwin, 44, was killed 4 September when the barb from a stingray pierced his chest while he was filming a TV show about the Great Barrier Reef. His family held a private funeral service 9 September.
His death set off an outpouring of grief, leading tens of thousands of people to travel to the 60-acre zoo near Brisbane to drop off flowers and other mementoes, many of them signing Irwin-styled khaki shirts instead of a condolence book.
There has been a surge of donations to the Irwin-instituted charity Wildlife Warriors, and millions of people have visited his website to find out more about Irwin and his conservation efforts.
Irwin's widow, Terri, made her first public appearance since his death at Wednesday's memorial service, as she thanked supporters and requested many of the tributes be featured in the service so those who missed out on attending could be there "in spirit". The couple's daughter, Bindi, eight, and son Bob, two, accompanied their mother.
"My daddy was my hero, he was always there for me when I needed him," said Bindi.
"We filmed together, caught crocodiles together and loved being in the bush together. I want to help endangered wildlife just like he did."
"Please don't grieve for Steve," his father Bob told the service. "He's at peace now, but I'd like you to grieve for the animals, for the animals have lost the best friend they ever had, and so have I."
John Stainton, Irwin's manager and close friend, said the service was to be a celebration of the naturalist's life.
"There will be one seat alongside of the family for Steve because he loved the Crocoseum, he built it," Stainton said prior to the service. "And his Australia Zoo cap that he always wore watching all the shows with his daughter will be on the seat."
The service included visual tributes to Irwin, with some "memorable film clips" and "funny moments" from his television career. Actor Russell Crowe was reportedly among those paying tribute by video.
"The one thing I hope everyone will take away from it is what a diverse character he was," Stainton said.
Taped video tributes were played at the service from Oscar-winning actor Russell Crowe, Kevin Costner, Cameron Diaz and singer Justin Timberlake.
Australian Prime Minister John Howard attended the service, saying Irwin touched the hearts of millions around the world.
In a final tribute, Australia Zoo staff spelled out Irwin's catchphrase "Crikey" in yellow flowers as his truck was driven from the Crocoseum for the last time to end the service.
