The crowd, nearly three times the previous record of 14,000 set in 2008, saw leaders Juventus beat second-placed Fiorentina 1-0 with a late goal from Sofie Junge Pedersen to put them on the brink of winning the title.
Tickets were free although they had to be reserved beforehand.
Defending champions Juventus are four points clear at the top of the 12-team table with three matches to play.
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A women's Serie A was first established in Italy in 1968 although it was only brought under the control of the Italian Football Federation (FIGC) this season.
"Taking the lead and winning in front of all these people is emotional," said Juventus coach Rita Guarino.
"I look back and I think of all the people who fought with me to make this sport more and more appreciated and followed: this was really an important day."
Juventus made their debut in women's Serie A last season and won the title at the first attempt.
(Writing by Brian Homewood; Editing by Ian Ransom)
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