Burnley finally surrendered their proud 17-game unbeaten start to the season despite a stirring second half fightback at Ewood Park.
The Clarets had made the short journey down the M65 hoping to keep the momentum going and build on two draws in their opening couple of Premier League cup games.
However, an experienced Rovers side ultimately made Burnley pay for missed chances to maintain their own 100 per cent record in the competition.
Blackburn fielded a team with a collective 331 first team games played, including striker Ben Brereton, a £7m signing from Nottingham Forest.
And goals either side of half time from Dominic Samuel and Jacob Davenport looked like setting Rovers on the way to a third straight group victory.
Burnley had other ideas though, and after Harker halved the deficit midway through the second half, Andy Farrell’s side laid siege to the Blackburn goal, only to be denied a share of the spoils by two stunning saves from Andy Fisher.
The Clarets settled into the game well, and following a crunching Adam Phillips challenge on Harry Chapman, Scott Wilson soon headed over from the Clarets’ first corner of the afternoon.
There was then a claim for a penalty 17 minutes in when Harker went to ground under a challenge from Lewis Thompson as the striker cut into the box from the right.
However, referee Simon Mather was unimpressed and waved away the appeals.
And Burnley spurned a golden chance to grab the lead soon after.
Harker pounced on a woeful back pass and saw his initial shot half-cleared by Fisher.
The loose ball spun to Josh Benson 15 yards out and with the ‘keeper stranded, his first-time effort was hacked off the line by last man Hayden Carter.
And they were punished for that double miss just after the half hour mark, when a superb strike from Samuel broke the deadlock.
The Rovers wide-man found space 30 yards out, and following a drop of the shoulder, unleashed a rising drive that gave Adam Legzdins in goal no chance.
The Clarets responded and Benson’s effort from outside the area was comfortably tipped over by Fisher.
But the Blackburn keeper pulled off an even better save five minutes before the break, somehow gloving Benson’s cleverly-disguised free kick around the post.
A frantic start to the second half saw Legzdins underline his credentials with a fine full-length save low to his right to deny Samuel.
But the second goal arrived soon after to kill off hopes of a Clarets revival.
Phillips’ stray pass in the centre circle saw the hosts counter and from Brereton’s stabbed cross, Davenport looped home from 12 yards.
Harker restored hope for the Clarets in the 69th minute, turning well in the box and finding the bottom corner to reduce the arrears.
And with Rovers suddenly rattled, Phillips had a glorious chance to level things up with 15 minutes remaining, when he robbed the last defender of possession 30 yards from goal.
However, with just Fisher to beat, the hosts’ last line of defence made yet another superb save to deny the Clarets midfielder.
The same combination brought the same end result 10 minutes from time, when Phillips thought he had squeezed a 30-yard free kick into the bottom corner, only for Fisher to again perform heroics with a fingertip save.
And despite Burnley throwing bodies forward in support of a three-man attack in the closing stages, Rovers held on to grind out the victory.
BURNLEY: Legzdins, Cooney, Glennon, Phillips, Younger, Wilson, Koiki, N’Guessan (Richardson 86), Harker, Mumbongo (Thomas 59), Benson (Goodridge 80).
Subs: Jensen, Tucker
BLACKBURN: Fisher, Mols, Thompson, Davenport, Whitehall, Carter, Samuel (Vale 71), Gladwin (Paton 45), Brereton (Butterworth 60), Rankin-Costello, Chapman
Ref: S Mather