The country music legend, 76, joked that she would ‘rather have a kidney stone’ then become political, but asked viewers to send ‘love and hope’ to those affected by the conflict.
Speaking before the first announcements of the winners, the singer asked the crowd to “take a serious moment” before the start of the show.
“Now I don’t want to do politics and that’s not the case. I’d rather pass a kidney stone than do this,” she said.
“I want us to send our love and hope to our brothers and sisters in Ukraine.
“So why don’t we dedicate this whole show to them and pray for peace in this crazy old world.”
The show took place in front of a large crowd at Nevada’s Allegiant Stadium.
The ACM Awards, which are widely considered country music’s biggest night, included performances from Kelly Clarkson, the Osbourne brothers and Parton herself.
Parton wore a sparkly jumpsuit with a sparkle design and joked that a disco ball fell on her before the show.
“I wanted to shine tonight, did I succeed?” she asked the audience, to cheers and applause.
Kelly Clarkson took the stage to perform Whitney Houston’s I Will Always Love You, which she dedicated to Parton.
The biggest prize of the night, Entertainer of the Year, went to Miranda Lambert, who accepted the award virtually from London.
Male Artist of the Year went to Tennessee Whiskey singer Chris Stapleton, while Best Female was won by Carley Pearce.
Morgan Wallen won album of the year at the Dangerous: The Double Album awards, a year after being dropped from the ACM ballot after being caught on camera using a racial slur.
He was also suspended by his label Big Loud Records in February 2021 after footage surfaced of him shouting insults outside a house in Nashville.
The singer made no direct reference to the incident when accepting the award, but said the album would signify to his son that “dad was a fighter”.