The regional airline wants the Ministry of Defence to reconsider its application.
It plans to operate twice-daily return services to an initial five UK regional destinations from the London airport.
Northolt currently is used mainly for business flights.
Many regional airports are worried that they will lose their London links as airports opt for more lucrative long-haul flights.
Saad Hammad, Flybe’s chief executive, said: “Northolt has, for too long, been the preserve of the elite with their private jets.
"The government is in danger of turning its back on an opportunity that would benefit hundreds of thousands of people in giving immediate regional air connections to London and beyond through Heathrow’s long-haul network."
He added: "Opening-up Northolt would provide many of the domestic benefits of additional runway capacity now without any additional noise impact.”