For the first time since 2018, Serena and sister Venus (42) revived the partnership which has brought them 14 doubles titles at the majors.
However, their hopes of collecting a third crown in New York were dashed in a 7-6 (7/5), 6-4 defeat to Czech pairing Linda Noskova (17) and Lucie Hradecka (37).
The loss leaves 40-year-old Serena free to focus on what is widely expected to be the final singles campaign of her landmark 27-year career before retirement.
On Friday, the 23-time Slam singles winner will face Australia's Ajla Tomljanovic (46) for a place in the fourth round.
Serena and her 42-year-old sister went down fighting, clawing back from 1-4 down in the second set before the Czech pair steadied the ship.
"Im still in shock that we won because this is the first time we played together," said Hradecka.
"I'm so sorry for you that we beat them but we are so happy that we did it."
Meanwhile, Azarenka (33) triumphed against Marta Kostyuk (20) 6-2, 6-3 in a match dominated by bitter exchanges over the conflict in Ukraine.
Last week, Belarusian star Azarenka was axed from an exhibition event aimed at raising money for the war-ravaged country.
Ukraine's Kostyuk had refused to take part in protest at the presence of Azarenka who she has criticised for a perceived failure to condemn the invasion of her home country.
Belarus is a close ally of Russia and has allowed Moscow to use its territory to launch attacks into Ukraine.
The tension between the two was evident at the end of their tie on Court 17 with Kostyuk offering just a touch of racquets instead of the customary post-match handshake.
"How did it make me feel? It's not the most important thing in the world right now," said Azarenka.
- No handshake -
Kostyuk, the 20-year-old world number 65, said a handshake would not have been appropriate.
"I genuinely wanted to warn her that I'm not going to shake her hand," she said.
"She never came up to me personally to tell me her opinion, what she thinks (about the war)."
Away from the controversy, world number one and French Open champion Iga Swiatek (21) cruised into the third round with a 6-3, 6-2 win over 2017 champion Sloane Stephens (29).
Paula Badosa (24), the fourth seed and potential semi-final opponent of Swiatek, suffered another miserable return to the city of her birth.
The Spaniard's failure to get beyond the second round will stretch for another year after losing 6-7 (5/7), 6-1, 6-2 to Petra Martic (31) of Croatia.
Sixth seed Aryna Sabalenka (24), a semi-finalist in 2021, came back from 1-5 down in the second set and saved two match points to defeat Kaia Kanepi (37), 2-6, 7-6 (10/8), 6-4.
China will have four women in the last 32 for the first time at a Slam after Zheng Qinwen (19) defeated Anastasia Potapova (21) and Yuan Yue (23) got the better of Irina-Camelia Begu (32).
Zhang Shuai and Wang Xiyu had already booked their spots in the next stage.
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