Former race car driver, who lost his legs in a Champ Car accident in 2001, suffered head injuries after being hit by a lorry.
Zanardi suffered severe facial and head injuries when he crashed into the heavy vehicle on a descent during the "Obviettivo Tricolore", which sees Paralympic athletes ride across the country in handbikes, cycles, or racing wheelchairs.
The 53-year-old was transferred by helicopter to the Santa Maria alle Scotte hospital in Siena on Friday evening.
"He was immediately taken care of by the emergency room professionals, assessed in the shock room and his condition is very serious due to the severe head injury," the hospital said.
"The athlete began neurosurgical and facial intervention for the serious head injury shortly after 7pm and this ended at 9.30pm."
Zanardi, a former Formula One and Champ Car race driver, lost both his legs in a Champ Car accident in 2001.
He made a successful return to top-level racing in an adapted car and competed in the World Touring Car Championship after his accident.
The Italian then switched his focus to hand-cycling, winning two golds at the Brands Hatch race circuit which hosted the London 2012 Paralympic cycling events, before winning two more golds in Rio.
Italy's prime minister, Giuseppe Conte, sent his best wishes.
"You never gave up and with your extraordinary strength of mind you have overcome a thousand difficulties," he wrote on Twitter.
"Come on Alex Zanardi, do not give up. All of Italy struggles with you."
His former F1 team, Williams Racing, also paid tribute to him on Twitter.