The painting, which was recently found in a private townhouse in Paris, sold for 2.3 million euros ($A4.1 million) on Sunday.
It depicts the crucifixion of Jesus Christ.
It was part of a French collection and was initially thought to be from one of the many Rubens workshops that existed at the time.
The artwork was rarely valued at more than 10,000 euros.
"I immediately had a hunch about this painting, and I did everything I could to try to have it authenticated," auctioneer Jean-Pierre Osenat told the Associated Press.
"And finally, we managed to have it authenticated by the Rubenianum, which is the Rubens committee in Antwerp."
Nils Büttner, an expert known for his research on Rubens, explained before the auction that the master often painted crucifixions but rarely depicted "the crucified Christ as a dead body on the cross".
"So this is the one and only painting showing blood and water coming out of the side wound of Christ, and this is something that Rubens only painted once."
The Osenat auction house said the painting's authenticity and provenance were confirmed after scientific analysis.
It said microscopic examination of the paint layers revealed not only white, black and red pigments in the areas representing flesh but also blue and green pigments, whose use is typical in Rubens' depictions of human skin.
Art expert Eric Turquin told a packed house the painting had virtually disappeared in the early 1600s.
It is known to have belonged to 19th-century French classic painter William Bouguereau before it was passed down in the family.
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