The one-day star has failed to impress in the first four matches of the series, either as an opener or when he dropped down the order last time out at Old Trafford, where he made a top score of 31.
Craig Overton has also been left out for the clash at the Oval, with Sam Curran and Chris Woakes, who made way for Overton at Old Trafford, taking their places.
The England and Wales Cricket Board also confirmed Ben Stokes would not bowl in the match, which gets under way on Thursday, due to a shoulder injury, and will play as a specialist batsman.
England need a victory at the Oval to draw the series, although Australia have already retained the Ashes.
Captain Joe Root said that Roy had missed out due to Stokes’ injury, which meant the side needed re-balancing.
“It’s always tough to leave guys out, but Stokesy obviously picked up a shoulder injury in the last game and won’t be able to bowl the overs we normally expect him to,” Root said at a press conference.
“With that we’ve had to change the balance of the side, and Jason’s the unfortunate one to miss out.
“Jason’s had an opportunity to come in and play Test cricket, get a feel for it and it’s not quite gone how he would have liked.
“But I’m sure he’ll go away and work extremely hard and come back again.
“That’s what you expect of guys when they get left out and I’m sure he’ll have that attitude and will want to try and prove a point and get himself back into the side.”
Root, who said he had spoken to Roy about his omission, felt it would have been a risk to include Stokes as a fourth seam bowler.
“He might be able to bowl a couple of overs here and there, but his batting, as this series has shown, is a massive part of our side and he rightly deserves to be in this team as just a batter, and a top-four batter,” Root said.
“So he will I’m sure want to find different ways of getting himself into the game, even if it’s not with the ball.”
Root insisted there was plenty still for his side to play for at the Oval.
“It’s bitterly disappointing not to have got the Ashes back, but we haven’t lost anything yet,” he added. “We’ve got to make sure we square the series up.
“And there’s the Test Championship points to play for as well, which down the line could make a huge difference, so there is plenty to play for.
“The guys are fully aware of that and very determined to make sure we finish the series 2-2.”