The petition had been organised by a campaign group following the death of Dr John Hinds in July.
Dr Hinds was known as one of the "flying doctors" of Irish road racing, providing medical cover at the events.
Earlier this year he had called for the provision of an air ambulance for Northern Ireland.
More than 65,000 people have given their support to the petition online, and 16,000 more have signed on paper.
Traditional Unionist Voice leader Jim Allister presented the petition in the Northern Ireland Assembly on Tuesday.
He said that the presentation was "tinged with sadness" because the "great champion of this cause is no longer with us".
"It was Dr John's vision and his passion for the need to deliver a proper air ambulance service to Northern Ireland that started this campaign [and] invigorated it," Mr Allister added.
Last week, Stormont's Health Minister Simon Hamilton said he was committed to establishing an emergency medical helicopter service in Northern Ireland as part of a new trauma network.
The minister added that because of the difficult financial climate, his department would "need to establish if it is possible to secure a robust and recurrent charitable funding contribution for this service".
Mr Allister told the assembly the Department of Health should "show its bona fides" by "capital funding the acquisition and the equipping of the helicopter".
A petition signed by more than 80,000 people calling for an air ambulance service to be established has been presented to Stormont.