The Florida club, who have built up strong support during a successful period playing at minor-league level, will join the top division for the 2015 season as its 21st team.
MLS commissioner Don Garber said: "We are proud and excited to welcome Orlando to Major League Soccer.
"From the success of the Orlando City Soccer Club, to the incredible fan support and passion in the community, this is a new market that really excites us."
The group that owns the club, which moved to Orlando from Texas three years ago, is headed up by Brazilian Flavio Augusto da Silva and also includes Phil Rawlins, a director at English Premier League side Stoke.
Rawlins said: "It is a dream come true to bring Major League Soccer to Orlando.
"The fans in Orlando have clearly demonstrated that they are ready for MLS, and we look forward to celebrating with them and working together to set a new standard in this country.
"We have watched the evolution of Major League Soccer, and we are thrilled to join what has become one of the fastest growing and most exciting leagues in the world."
Building work on a new 18,000-seater stadium is set to begin in the spring, though it is not scheduled to be ready in time for the start of the Lions' first big-league season.
Also joining MLS in 2015 will be the league's 20th franchise, New York City.