The 134-mile route, which is named after the 19th Century Scottish conservationist, launched in 2014 to mark the centenary of his death.
It runs between Helensburgh in the west and Muir's birthplace in Dunbar.
The John Muir Way is the 28th long distance route to be given the accolade by Scottish Natural Heritage.
The route, which includes castles, towns, villages and Loch Lomond and The Trossachs National Park, attracted more than 60,000 people in 2015.
Muir left Scotland for America aged 11 and became one of the most influential figures in the history of the environmental movement.
Keith Geddes, chairman of the Central Scotland Green Network Trust said: "There is a growing understanding in Scotland of Muir's significance throughout the world and the award of Scotland's Great Trail status will help to further raise awareness of both Muir and the route.
"The John Muir Way has proved to be popular with locals going out for a short walk, for those long distance walkers who complete the route on consecutive days or over several visits and to hundreds of visitors from abroad, particularly from Muir's adopted homeland wanting to learn more about Muir's life in Dunbar."
Scottish Natural Heritage chairman Ian Ross, said: "The coast to coast route is the second longest of our Great Trails and, spanning central Scotland, it is easily accessible for millions of people living here.
"The John Muir Way is also another excellent outdoor tourism asset for Scotland and we hope the award helps to attract many more visitors over the coming years."
The John Muir Way has been awarded Scotland's Great Trail status three years after the coast-to-coast route was established.