The 34-year-old Bath hooker aggravated a shoulder injury during Saturday's 25-24 defeat by Ireland in the Millennium Stadium.
He has been forced to pull out of the squad for the trip to the Stade de France, almost certainly ending his international career.
Wales team manager Alan Phillips said: "He has not received from the injury and although we are still awaiting the report from the specialist, Jonathan will not be considered for Saturday.
"I feel for him because since he has come into the side, he has been inspirational. His contribution has been outstanding, I can't say anymore."
Humphreys is likely to confirm his retirement from international rugby after this weekend.
He returned to the Wales fold after almost four years in exile following the call from coach Steve Hansen after injuries to Robin McBryde, Andrew Lewis and Barry Williams left Wales short on front-row forwards prior to the England game.
However, better news for Hansen is that Bridgend hooker Gareth Williams, who broke a bone in his hand against Scotland 17 days ago, is back with the party and likely to take over in the 22 from the inspirational Humphreys.
Pontypridd's Mefin Davies, a second-half replacement for Humphreys on Saturday, is likely to start with Williams on the bench.
Winless Wales must take the points in Paris or they will end the tournament with the dreaded Wooden spoon.