Klaire Holloway claimed in a UK newspaper on Sunday that she had a one night stand with Charley’s ex after they met at a family barbecue.
But Charley, 25, has dismissed the allegations, saying there is no bad blood between her 33-year-old co-star - who she split from months ago and has three-year-old son Buster with.
She said: 'Me and Matthew are great friends. I know what the truth is and I know people who were there. There are a lot of witnesses.
'Matthew is Buster's dad and we're both really great friends.'
The Emmerdale stars - who play Debbie Dingle and David Metcalfe in the ITV soap - got engaged in 2009 but announced their split in August.
A spokeswoman for the show said at the time: 'Charley Webb and Matthew Wolfenden have confirmed that they have separated. They will remain friends and are focusing on their little boy.'
Matthew thanked fans on Twitter after ending his five year relationship.
He wrote: 'Thank you so much for all your lovely messages. Unfortunately these things happen, and it's very sad. Buster is our main priority always. X.'
Meanwhile, Charley has also confirmed her soap star sibling Jamie Lomas is ‘fine’ after allegations his wife Kym cheated on him with her Coronation Street co-star Oliver Mellor.
Speaking at the Inside Soap Awards on Monday, Charley admitted she is 'very proud' of her brother for 'remaining quiet.’
She said of the EastEnders star: 'We're very close and at the end of the day he's fine and he's got his dignity and that is all that matters.
'He's remained quiet and I'm very proud of him. We've both been in the business for a long time.'