After just 67 days in charge, Rovers jettison the man who was already their fifth manager of the season, with the club sitting 18th in the Championship after drawing with Burnley
The 37-year-old was already Rovers' fifth boss of the season, having joined from Blackpool after a similarly short-lived spell at Bloomfield Road.
Appleton replaced Henning Berg in the Ewood Park hot seat on January 12 but has himself been dismissed following a poor run of results.
A statement from the club's official website says: "Blackburn Rovers FC can confirm that Michael Appleton has been relieved of his duties as manager along with assistant manager Ashley Westwood, first team coach Darren Moore and head of senior recruitment Luke Dowling."
Appleton recorded victories in four of his first seven games, including knocking Arsenal out of the FA Cup earlier this season, but Rovers have only won four out of their last 15 games.
Following Sunday's 1-1 draw with bitter rivals Burnley, Blackburn are 18th in the Championship table, four points clear of the drop-zone in their first season since relegation from the Premier League.
Reserve team coach Gary Bowyer is the bookmakers' favourite to be put in charge for the remainder of the season.
Appleton began the season in charge of struggling League One club Portsmouth, before moving to Blackpool in November. He only lasted at Bloomfield Road for two months before switching to the Seasiders' Lancashire rivals.