A flight departed Dubai about 9.30am AEDT, and is scheduled to land in Sydney on Wednesday about 10.30pm.
It will be the first commercial flight to arrive in Australia from the region since the closure of airspace due to the outbreak of the conflict.
Foreign Minister Penny Wong said six crisis teams were being sent to the Middle East to provide extra consular support to trapped Australians, but would not confirm their locations due to security reasons.
Iran has launched retaliatory attacks on 10 countries in the region following US-Israel assaults.
There are about 115,000 Australians in the region, with 24,000 in the United Arab Emirates.
Senator Wong said there had been an "unprecedented number of registrations" as the government assessed their eligibility.
The foreign minister said the government was not considering sending Australian troops to the Middle East and repeated that commercial flights remained the best way to get large numbers of people back to Australia.
Prime Minister Anthony Albanese urged people to stay in contact with their airlines about their options.
"We understand this is a very distressing and challenging time for Australians who've been caught up in these events," he told parliament.
Mr Albanese spoke with the president of the UAE, Mohammed bin Zayad al Nahyan, on Tuesday night and thanked him for the hospitality shown to Australians stuck in transit.
The prime minister also spoke with his New Zealand counterpart Christopher Luxon about consular issues arising from the war, which began when the US and Israel began hitting Iranian targets.
United Nations special rapporteur Ben Saul said it was "crystal clear" the attackers' actions were an unlawful, armed aggression against Iran.
"These aren't acts in self-defence because Iran has not attacked either country and is not about to imminently attack them and the security council hasn't given any authorisation," he said.
Professor Saul accused the Australian government of "trashing" the rules-based world order while it dodged questions about the legality of the attacks.
Australia was one of the first countries to back the strikes by US and Israeli forces over the weekend, although government ministers have said it was up to the instigators to argue the legal basis for their military action.
Tehran has retaliated with a barrage of drones and missiles aimed at neighbouring states, targeting oil and natural gas infrastructure.
Australia's embassy in Saudi Arabia has warned citizens in Riyadh, Jeddah and Dhahran to shelter in place, while a defence base in the UAE housing Australian troops was previously hit in an Iranian attack.
Opposition foreign spokesman Ted O'Brien said Labor should be looking at every option to ensure stranded Australians were kept safe.
"We need to be working, in particular with our allies and partners, many of whom are taking more proactive steps to get their own citizens out," he told Sky News.
"The government needs to be leveraging those partnerships as much as it can."
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