The service has the aim to transport Catholic pilgrims to holy sites throughout the world, and its first flight will take pilgrims from Rome to Lourdes in France on Monday.
The first scheduled flight is set to depart at 0930 GMT and will use a small charter airline owned by the Italian post office.
The airline is targeting to transport up to 150,000 pilgrims within its first year of operation.
Initial flights have been scheduled for Catholic holy sites in France, Poland, Spain as well as the Middle East, with the future goal of including Mexico in the near future.
The Vatican has signed a five-year agreement through its local travel organisation which organises pilgrimages for the Diocese of Rome.
The charter airline, called Mistral Air, normally carries courier cargo, letters and parcels for the Italian post office.
The Vatican itself has no airline and no airport - just a helipad used by the Pope for local journeys with the privilege of the Italian air force providing helicopter transport.
Mistral Air will commence service with two charter jets and they will be carry the Vatican colours of yellow and white.
Reports have indicated that the interiors of the planes have been decorated with sacred inscriptions such as "I search for your face, Lord" hoping to inspire pilgrims on their travels.
