"@WayneRooney misses out on the final #mutour match due to a shoulder injury sustained at the weekend," United said in a Tweet.
David Moyes said: "It’s not too serious".
The match is United's final tour match before competitive action begins with the Community Shield clash against Wigan next weekend.
It represents something of an embarrassment for the Premier League champions, who used Rooney's image to promote the match against AIK in Stockholm - a match that can only be watched in the stadium or pay-per-view on the club's website.
Rooney, 27, was the subject of an unsuccessful transfer offer from the Blues in July, days after he was pulled from United’s Far East Tour.
Back then Rooney was disillusioned after being described as a squad player by new United boss David Moyes.
This time he was pulled from the pre-season friendly against AIK Stockholm in Sweden, with the club citing a shoulder injury suffered in a behind-closed-doors friendly with Real Betis at the weekend.
Rooney’s withdrawal comes hours after United confirmed a second Chelsea bid - believed to be in the region of £25-30m - had been received and rejected, and the ‘injury’ will add fuel to the speculation that the England striker wishes to force a transfer.
A United spokesman earlier told Press Association Sport: "A bid was received yesterday and immediately rejected. Our position remains that he is not for sale."
Rooney, 27, has been linked with a move away from Old Trafford after falling behind Robin van Persie in the pecking order.
Chelsea have been most closely linked with the former Everton man, having seen an offer dismissed earlier this summer.
It is reported their latest bid was £25 million plus add-ons that would push the deal to the £30m mark, but that has not proved enough to tempt David Moyes.
Rooney has scored nearly 200 goals for Manchester United after joining from boyhood club Everton in 2004.
But his time at Old Trafford has been inconsistent, with form and fitness up and down for the burly centre-forward.
Rooney backed down after requesting a transfer in 2010, but he had a far stronger hand back then, having scored 26 Premier League goals the previous season.
The next campaign was not one of his best, but he reached a career-record 27 league goals the following campaign as United finished second to Manchester City.
The signing of Netherlands striker Van Persie saw Rooney in and out of last term's title-winning side, with since-retired boss Sir Alex Ferguson claiming a second transfer request was submitted late in the season.
New boss Moyes appeared to hint Rooney was no longer first choice, saying he was needed at the club in case of an injury to Van Persie, but he later backtracked and insisted the player was not for sale.
Chelsea reportedly initially offered £20m but that was swiftly rejected; while the latest bid was also turned away, United's resolve will surely be tested for a player that many feel wants a new challenge.
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- David Moyes