The six-time US Open champion lost 7-5, 6-7 (4-7), 6-1 to Ajla Tomljanovic but the game was nothing short of exciting as Serena gave it her all before succumbing to her Australian opponent.
As usual, the 40-year-old received a rousing welcome from the home crowd who once again flocked in huge numbers to witness Serena in what could possibly have been her final US Open appearance.
Serena began on a positive note, leading 3-2 in the first set but Ajla showed excellent grit to come from behind and win it 5-7.
Despite losing the first set, Serena found constant support from the crowd, especially her husband Alexis Ohanian and family members who were close to her in the stands.
This worked in Serena's favour as she upped her game to take the second set and keep herself in the contest.
However in the third set, Serena looked a little tired and committed few errors which cost her the game.
Ajla, on the other hand, showed nerves of steel to counter the champion star who clearly had the whole crowd's backing.
One of the best moments of the match happened in the final minutes as Serena saved five match points at 5-1 down before her final shot hit the net, thus, putting a halt to her US Open 2022 campaign.
Following her loss, the US star shook hands with her 46th-ranked opponent and made sure she showed her appreciation to the crowd by thanking them.