The Council of State cited the "long-standing decline" of the San Lorenzo market as the reason for its decision, upholding a previous decision from a regional court to move around 80 stalls out of the square.
Florence's tourist office describes the market as "a food, leather, and cultural adventure", and street traders had lodged an appeal to keep it open. They argued that the historical market was subject to protection as a cultural site within Florence's historic centre, which is a Unesco World Heritage site.
But judges disagreed, saying that "over the years, the evolution of the market showed signs of degradation, resulting in the loss of many typical characteristics of the original historic market," in the ruling published on Wednesday.
Tax evasion by stall traders and the selling of counterfeit goods were two of the main problems listed, as well as the need to make the San Lorenzo basilica fully accessible to worshipers and tourists.
The court said that the market had become "incongruous and inconsistent" with the historic and artistic character of the city centre, and that the removal of its stalls was part of an ongoing project to "upgrade the historic San Lorenzo quarter; one of the most important and central complexes of this city of art".
On travel review site TripAdvisor, the market is ranked as the seventh most popular activity in the Tuscan capital.
Plenty of tourists labelled it a "must do" and praised the authenticity and prices of the souvenirs for sale - though others said the indoor food market was the highlight, with some complaining that the outdoor stalls were repetitive and overpriced.
"Several years back when we were here, the quality of goods was better and good value," wrote one reviewer from Greece. "There is less variety these days."
The Tuscan capital is fiercely protective of its heritage, and its mayor last year rejected a request from McDonald's to open a chain in the city's central square. After social media protests and a petition from residents, mayor Dario Nardella turned down the application, saying he wanted to support "traditional businesses" in the city.
Some months earlier, the city passed a law aimed at ensuring that at least 70 percent of produce in all new eateries was locally sourced, amid worries that cheap kebab shops and other fast food outlets meant the city was at risk of losing its character.