Appleton's sacking after just 67 days was confirmed in a short club statement. His predecessor Henning Berg managed 10 days less at the helm before he too was fired.
Indian poultry firm Venky's are now looking for a fifth permanent manager since their £23million takeover of the club in November 2010. Sam Allardyce was the first boss to go, sacked within a month of the buy-out with the club 13th in the Barclays Premier League.
He was succeeded by Steve Kean, who resigned last September following a long campaign of protests from supporters against him.
Rovers are currently 18th in the npower Championship, only four points above the relegation zone.
Wayne Wild, the co-chairman of supporters group the Rovers Trust, said in a statement: "News of Michael Appleton's sacking ought to come as a surprise, but sadly it does not.
"This is just the latest in a long line of incomprehensible and ill-thought out decisions that go back to the very start of the Venky's ownership of Blackburn.
"It is obvious to any experienced fan or football professional that to keep changing managers - we are now looking for our sixth this season - will only lead to yet more instability and uncertainty. It is also a costly exercise to keep paying off staff.
"The owners have to face up to the fact they are entirely responsible for the situation we find ourselves in with a second successive relegation a distinct possibility."
- Sports & Recreation