The ad, produced by DTV, forms part of the charity's 'Cut Them Free' campaign to stop child sexual exploitation, a growing problem online. Media was handled by MC&C.
Focusing on the dangers of grooming, the ad will run for the first time during on 3 October at 9.24am, on Comedy Central during 'Two and a Half Men'.
The ad features a man, shot in darkness to hide his face, talking about how he grooms children online. The ad then cuts to a young girl on her laptop and shows her going to meet her new friend, before she realises he is actually a man.
The ad asks viewers to donate £3 a month and will also run on digital channels across the UK and includes two test two formats.
Martin Field, fundraising director at Barnardo's, said: "The thinking behind the advert is to alert people to the importance of the work we do in highlighting the risks to children of sexual exploitation and protecting them.
"We want to warn parents that their children's friends aren't always what they seem and we want to touch people's hearts and make our cause their cause."
Barnardo's works with more than 1,000 sexually exploited children across the UK each year.
Follow Sara Kimberley on Twitter @SaraKimberley