The Gunners face Bayern Munich in the Champions League on Tuesday night and manager Arsene Wenger had made several changes, with Jack Wilshere, Theo Walcott and Santi Cazorla all starting on the bench.
Gervinho missed a great chance at the end of the first half which proved costly as Colin Kazim-Richards' scrappy effort after 72 minutes was enough to inflict Arsenal's first home FA Cup defeat since their move to the Emirates Stadium.
Blackburn, who still have not conceded a goal in this season's FA Cup, fashioned an early chance when a free-kick from the left was flashed through the Arsenal six-yard box and Markus Olsson knocked the loose ball wide at the far post.
In the 25th minute, Arsenal finally got the delivery right from Tomas Rosicky, and Abou Diaby rose to power a bullet header goalwards, which Rovers goalkeeper Jake Kean beat away at full stretch with a magnificent reaction save.
The best opening came just before half-time when Rosicky slipped Gervinho into the penalty area, but despite holding off the defender, the Ivory Coast forward dragged his shot wide of the post - before dropping to his knees and looking to the Heavens.
Rosicky crashed a 20-yard effort off the crossbar and Rovers defender Grant Hanley made an important block as Gervinho cut in from the right.
With 20 minutes left, Wenger had seen enough and made a triple substitution as Walcott, Wilshere and Cazorla replaced Gervinho, Rosicky and Alex Oxlade-Chamberlain.
However, out of nothing, Blackburn snatched the lead. Martin Olsson burst into the left side of the penalty area and drilled the ball goalwards, which Wojciech Szczesny parried. Kazim-Richards' scuffed follow-up effort bounced onto the post and in.
Diaby's effort was charged down and Kean somehow saved a close-range header from Walcott before Olivier Giroud's effort in stoppage-time was blocked as Rovers clung on for a famous win.