The sisters decided to give it one last whirl four years after their last slam appearance together and six years on from the most recent of their 14 titles.
But the old magic was not quite there and they fell to a 7-6 (5) 6-4 loss to the Czech duo of 37-year-old Lucie Hradecka and 17-year-old Linda Noskova.
The match was surely the first opening-round doubles contest ever to begin a night session on Arthur Ashe Stadium.
When it was announced Serena and Venus would be playing doubles, it had appeared this might be the former's last match, but her success in singles gave this contest a slightly different complexion.
The competitive desire was very much there, and Serena again looked sharp both from the back of the court and at the net.
But Hradecka and Noskova hung on and then seized their chance in the tie-break, Hradecka threading a fine pass down the line on set point to virtual silence.
She was emerging as the key player against her fellow veterans and, although Serena and Venus retrieved an early break to get back to 4-4, a break of the Serena serve settled the contest.
Serena will now concentrate solely on trying to extend her final tournament in singles, where she will take on Australia's Ajla Tomljanovic in the third round on Friday.