The 45-year-old now has two Paralympic Games wins at the motor-racing circuit where he first competed as an F3000 driver 21 years ago.
Zanardi, who won men's Individual H4 Time Trial gold on Wednesday, won the 64km race in a sprint finish ahead of South Africa's Ernst van Dyk, with Belgium's Wim Decleir third.
'To win this race in this way, in such a dramatic way and sprinting makes me really proud,' said Zanardi, who is in action tomorrow in a third event, the H1-4 Team Relay.
'It shows that I am a complete cyclist, even if I have no legs. I just tried to save as much as I could during the race. I know I'm a good sprinter and just had to be confident in myself. And it worked.
'The race did not go as I expected. I thought that I could take off soon and that I would only need to stay calm, but at the fourth lap I tested my opponents and understood my tactic would not work, so I changed it completely and only tried to save, save and save.
'I used the uphills as rest, the others were overtaking me, then in the downhills I caught them up and regained my position.
'I managed my energies well. It's been a tough race, I could have very well lost it.'
I managed my energies well. It's been a tough race, I could have very well lost it.Alex Zanardi
The corresponding women's race was won by Andrea Eskau of Germany in dominant fashion.
The Netherlands' Laura de Vaan was 10:16 behind in second, winning a sprint for silver ahead of Germany's Dorothee Vieth, as Eskau added Individual H4 Road Race gold to the Individual H4 Time Trial title she claimed on Wednesday.
'I trained to drive on this track alone,' Eskau said. 'I divided up the laps. The first lap I really took it on, to get the right distance, and I did the timing that I'd been training on.
'The team tactic is that if one German is at the front, then the other shouldn't do that much work, and should focus on the sprint.'
Marianna Davis of the USA won the women's Individual H1-3 Road Race, adding to her victory against the clock in the Time Trial, with team-mate Monica Bascio 33 seconds behind to take silver.
Britain's two representatives crossed together, but Rachel Morris was awarded bronze as she and Karen Darke crossed the finish line holding hands in a failed bid to share third place.
Ireland's Mark Rohan won his second event of the Games with victory in the men's Individual H1 Road Race.
Rohan finished two seconds ahead of Tobias Fankhauser, with Wolfgang Schattauer of Austria third.
The men's Individual H3 Road Race was won by Rafal Wilk of Poland, another who also won Individual Time Trial gold.
Vico Merklein of Germany was second, with Joel Jeannot of France third.
Austria's Walter Ablinger claimed victory in the men's Individual H2 Road Race, Switzerland's Jean-Marc Berset was second and Italy's Vittorio Podesta third.