The TV star, 47, was pictured in what appeared to be a heated discussion with McPartlin, 42, ahead of the Manchester auditions for Britain’s Got Talent.
But Holden has since shut down any talk of a feud, claiming the auditions were filled with “too much laughter”.
The mother-of-two posted a picture of herself and McPartlin with Declan Donnelly and fellow judges Simon Cowell and David Walliams.
She captioned the shot: “I’m #loving #Manchester. Had a #fabulous #first #day up here – loads of fun. Too much #laughter @BGT.”
Holden – who is a judge on the ITV talent show – appeared to be shouting at McPartlin as fellow judge Alesha Dixon stood awkwardly alongside them at The Lowry Theatre in Salford on Thursday.
They were interrupted by a member of the Britain’s Got Talent production team who placed her hand on Holden’s arm before they took part in the photocall.
Her latest tweet comes hours after a source close to Holden claimed the pair were simply “chatting”.
Speaking to Mail Online they said: “This picture was taken whilst they were posing in front of hundreds of fans and press.
“There simply wasn’t any form of heated discussion whatsoever.”
A representative for McPartlin also shot down talk of an argument, branding it “absolute rubbish”.