Miller came on as a halftime substitute in Rovers' 3-1 English Championship win over QPR but just 10 minutes later, went down with no-one around him and had to be taken from the field on a stretcher.
Less than four months out from Australia's opening group stage match in Vancouver, the injury is a crushing blow for both Miller and the Socceroos, given the defender had established himself as starting right-back.
"It does look like a serious injury," new Blackburn head coach Michael O'Neill told Rovers club media.
"Obviously he's in a boot in there and we can only wish that it's not as serious as what it possibly looks.
"Obviously in a lot of pain and had to be stretchered off - and no-one near him, so obviously it looks like an achilles situation.
"He's got a big World Cup and everything ahead of him - so with all those things in the melting pot it makes it very, very difficult if the injury is severe.
"But we can only hope that over the next period he gets good news but at this time it's too early to say."
Miller's injury leaves Jason Geria - who only returned to club football with J2 League club Albirex Niigata last week - and Fran Karacic as the established right-backs fully fit and available to Tony Popovic ahead of send-off games in Sydney and Melbourne next month.
Jacob Italiano, who impressed in his two caps before suffering a calf injury, has only played a few minutes for Austrian club Grazer AK but will be right in the mix if fit, while centre-back Milos Degenek can play at right-back.
Miller is the first Socceroos regular to appear ruled out of the World Cup through injury.
Veteran Craig Goodwin last week suffered a groin injury that will sideline him for 10-12 weeks, putting his own audition for a place in the squad in peril, but is not ruled out.
Centre-back Harry Souttar has not played since his own ACL tear and is in a race against time to find fitness and form before Popovic picks his squad.
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