The 7-5 6-7 (7-4) 6-1 result brings a glittering career with 23 Grand Slams to a likely end.
It took over three hours for the gritty Australian to deliver the knock-out punch to Serena who was beginning to impress, having defeted second seed Anett Kontaveit in the second round.
Serena, 40, waved to all sides of Arthur Ashe Stadium while ‘Simply The Best’ played over the loudspeakers after the defeat as she walked to the net for a handshake.
A teary Serena while addressing the crowd said, “Thank you so much, you guys were amazing. I tried. Thank you Daddy, I know you’re watching. Thanks Mom.
“I just thank everyone that’s here, that’s been on my side for so many years, literally decades, But it all started with my parents and they deserve everything so I’m really grateful for them. And I wouldn’t be Serena if there wasn’t Venus, so thank you Venus. It’s been a fun ride.”
Asked whether there could be another chapter, Williams added: “I don’t think so, but you never know.”