The former Manchester United skipper was given his marching orders after a VAR review.
As the New York Red Bulls took a corner, Christian Casseres tussled with Rooney in the six-yard box and Rooney's arm struck Casseres's jaw.
The 33-year-old forward appeared to have escaped punishment after referee Ismail Elfath did not stop play, but the off-field officials reviewed the incident and Elfath was able to watch it again on pitchside monitors.
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The referee then sent off Rooney for the incident.
However, Rooney disputed whether he should have been sent off and after the game directed the blame to Elfath: “Ask the referee. I think anyone who knows the game of football can see what the intent was. It was to try to ease the player out.
“I'm disappointed because I got a red card and disappointed because I didn’t agree with the decision.”
Alejandro Romero had put NYRB ahead after just six minutes, and Rooney received his marching orders midway through the first half.
There was hope for the home side when Amro Tarek was sent off for New York Red Bulls at the end of the first half, and Ole Kamara equalised on 55 minutes.
Four minutes later Daniel Royer scored from the spot for the vistors. DC United now have six matches to play, with Rooney set to leave for Championship side Derby County in January.
Rooney's current side can no longer qualify for the MLS play-offs.