She went into the ninth and last stage with a lead of nearly four minutes over France's Juliette Labous and finished safely in the bunch in Olbia, northern Sardinia.
"This is the second one with Movistar, and I'm super proud of my team to finish it off," said the defending champion, who is due to retire at the end of this season.
Labous was runner-up while Italy's Gaia Realini finished third overall.
Van Vleuten, who also won the race in 2018 and 2019, became the Giro's oldest stage winner when she triumphed in the second stage aged 40 years and 266 days for her 100th career victory to take the maglia rosa.
The opening time trial scheduled for June 30 was cancelled due to heavy rain and thunderstorms.
"Now it's time to recharge the batteries and prepare for the Tour de France," Van Vleuten said.
The Tour de France Femmes will set off from Clermont Ferrand on July 23, the same day the men's race finishes in Paris.