The 32-year-old underwent a scan on the calf strain he suffered during Friday's 0-0 draw in Sweden on Sunday morning and the results have confirmed that he will be unable to play on Tuesday evening.
Wolves striker Kevin Doyle - informed by text that he had not made the original squad for the double-header - has been called up in place of Keane.
The news is a big blow for Ireland boss Giovanni Trapattoni, who had earlier said he expected the 32-year-old to be fit for what is a key game at the Aviva Stadium.
Trapattoni's team are currently fourth in Group C with seven points from four matches.
They are behind Austria on goal difference and one point behind second placed Sweden in the race for a play-off place.
Group leaders Germany already have 13 points and look certain to qualify automatically.
A FAI statement said: "The Football Association of Ireland today confirmed that Robbie Keane has been ruled out of Tuesday's FIFA World Cup qualifier against Austria in Aviva Stadium.
"A scan this morning confirmed that Robbie is suffering from calf strain after Friday's game against Sweden. Wolves striker Kevin Doyle has been called up in place of Keane.
"The manager has spoken to Kevin, and he will link up with the squad in time for training in Malahide tomorrow morning."
With Keane out, Trapattoni must now decide whether to start with Doyle - despite initially leaving him out of the squad - or Stoke's Jon Walters, who has been struggling with a back injury.
Derby's Conor Sammon and Nottingham Forest's Simon Cox could also come into Trapattoni's plans
Better news for Ireland is that midfielder Glenn Whelan, who missed the Sweden stalemate with an ankle problem, was able to train on Sunday and should be fit.