He was born in Palermo and was named after Vincenzo Florio, Sr. (1799–1886) who founded the Florio wine and spice company. In 1909 he married principessa Annina Alliata di Montereale. After her death to cholera in 1911, he married Lucie Henry of Épernay, France.
An automobile enthusiast, he also initiated the financial basis and the engineering plans for the Coppa Florio in 1900. His greatest fame came from creating the Targa Florio race in 1906, based on discussions with Gordon Bennett cup and Henri Desgrange.
Florio also raced, achieving 9th in a Mercedes-Benz 120 hp. His best result was the win in «Targa Rignano» (Padova - Bovolenta) 1903, named after Count Rignano, thus inspiring Florio to pick the surname-based Targa Florio. His own racing team, however, got Felice Nazzaro to leave Fiat and join as driver.
He died at Épernay.
Florio was also a painter. The exhibition Vincenzo Florio – A taste for modernity was on display in Palermo in 2003. The name has been commemorated in the Via Vincenzo Florio in Marsala, as well as the Vincenzo Florio Airport (1992) at Trapani.