Some charter flights currently fly between the countries but the new agreement could see as many as 110 flights a day - more than five times the current number.
The flights could begin in autumn this year.
This is the latest stage in a thawing of the countries' relationship.
US Transportation Secretary Anthony Foxx said the commercial flights marked a "critically important milestone in the US effort to engage with Cuba".
Cuban Minister of Transport Adel Rodriguez called it a "new era".
The US imposed a trade embargo on the communist-run island in 1960.
In late 2014, Presidents Barack Obama and Raul Castro announced that they would begin normalising their relationship.
They met for the first time in April 2015 in Panama, and shook hands and held a private meeting at the UN General Assembly in September.
There can be little doubt that the pace of the thaw between the United States and Cuba is quickening in President Obama's last year in office.
And this signing of a memorandum of understanding on commercial flights between the old enemies is a good example of that new impetus.
The announcement comes quick on the heels of a move by the US Treasury Department to allow the first US firm to set up premises in Cuba since Fidel Castro took over private business interests at the height of the Cold War.
Diplomatic relations between Washington and Havana have come a long way over the past year. Now they hope the economic links will follow fast.
Cuba reopened its embassy in Washington in July 2015 and a month later, the US reopened its embassy in Havana.
John Kerry visited the city for the ceremony and in doing so he became the first US secretary of state to visit Cuba in 70 years.
Mr Kerry has said the Obama administration wants to lift the trade embargo on the island, something Mr Castro says is essential for a normal relationship.
The Republican-controlled US Congress has blocked this.
The White House is contemplating a possible presidential trip to Cuba.
The US and Cuba have signed an agreement to resume commercial air traffic for the first time in more than 50 years.