Sayer had to be helped off the pitch while playing for Swedish club side Kristianstads DFF on the weekend, with scans confirming the severity of her injury.
Her ACL tear follows similar injuries to superstar captain Sam Kerr and young winger Holly McNamara.
Midfielder Katrina Gorry (ankle) and defender Clare Hunt (foot stress fracture) are racing the clock to prove their fitness before the Olympics.
Aivi Luik (hamstring), Emily Gielnik (calf) and Chloe Logarzo (hip) are also battling injuries.
Sayer, 22, has 11 caps and featured regularly in recent Matildas squads after just missing out on last year's Women's World Cup.
She scored her first goal during February's Olympic qualifiers against Uzbekistan and would have been pushing for selection for the 18-player squad for Paris.
"Since returning to the national team set-up in late 2022, Amy has been a positive addition to our team with her attitude and work ethic on and off the pitch," coach Tony Gustavsson said in a statement.
"To have suffered this setback is an upsetting blow for Amy and for our Matildas family.
"As always for us, our priority will be on supporting Amy and ensuring she is provided with care and assistance as she needs to navigate this challenging time."
Sayer will remain in Sweden for medical assessments before undergoing surgery then rehabilitation, with no timeline put on her return to play.
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