The Yemenia plane carrying 137 passengers, including patients needing treatment abroad and their relatives, took off from Sanaa on Monday for the Jordanian capital, Amman, just after 9am (06:00 GMT).
Before the takeoff, the plane taxied through an honour guard of two fire trucks spraying jets of water.
The flight was initially due to take off on April 24, but a dispute over passports issued by the Houthi rebels delayed it.
Last week Yemen’s government said it would allow citizens in rebel-held areas to travel on Houthi-issued passports, removing a barrier to the flights.
The government-run SABA news agency said last week that new Yemeni passports would be issued in Jordan for those arriving with Houthi-issued travel documents.
The conflict in Yemen, the Arab world’s poorest country, began in 2014, when Houthi rebels seized Sanaa. The Saudi-led coalition stepped in to support the government in 2015.
According to United Nations figures, more than 150,000 people have died in the violence and millions have been displaced, creating what it calls the world’s worst humanitarian crisis.
But a truce has been in place since April 2, coinciding with the start of the Muslim holy month of Ramadan.
Resuming flights from Sanaa, reopening roads to the rebel-besieged city of Taiz and allowing fuel tankers into the Houthi-held port of Hodeidah – a lifeline for Yemen – were all part of the agreement.
While fuel tankers have docked in Hodeidah and flights have now resumed from Sanaa, the main routes into Taiz remain cut off.
The inaugural flight under the truce had to be scrapped after Yemenia said it did not receive the necessary permits.
Each side blamed the other for the hold-up, while UN special envoy Hans Grundberg called for a quick resolution to the impasse.
The Norwegian Refugee Council (NRC) aid group said at the time that the inability to operate commercial flights out of Sanaa had stranded “tens of thousands of medical patients” seeking treatment abroad.
In a statement on Monday, NRC described the opening of the airport as a “stepping stone towards sustainable peace”.
“If the parties to the conflict continue to work together to operate regular flights in and out of Sanaa, they can help save thousands of lives, prevent premature deaths, and support the country’s economy,” said Erin Hutchinson, the aid group’s country director in Yemen.