The 46-year-old actress was questioned by David Letterman as to whether she'd be expanding her family.
"Have you thought about adding more to the group?," presenter David asked on his Late Show. "Oh sure," the American actress replied breezily.
David delved deeper asking, "Any chance?," to which Julia replied "No" with a smile.
Speculation began to swirl after the 46-year-old was photographed at the weekend wearing a loose-fitting jumper which seemed to be disguising a growing bump.
She was pictured at the BAFTA Britannia Awards last month wearing a floaty black mini dress which might also have hidden a blossoming baby bump.
The Pretty Woman star chose to ditch the heels for the glamorous occasion which saw her present a statuette to her fellow Hollywood leading light George Clooney.
Julia and her cameraman husband Danny Moder already have nine-year-old twins Hazel and Phinnaeus and six-year-old Henry who she calls the 'harbour of my life'.
The mother-of-three recently opened up about blissful family life.
"It's my privilege and an honour to cook three meals a day for my family, and it's a luxury on a level that I didn't even realize," the Oscar winning actress told Marie Claire.
Actress Julianne Nicholson, who plays Julia's sister in her upcoming movie August: Osage County, revealed more about Julia's domestic habits.
"I would go in the trailer, and she would be sewing sleeping bags for Hazel's dolls," she said.
While shooting the film, for which she received a Golden Globe nomination on Thursday, Julia made sure the "hours were such she can still be a mother" according to director John Wells.
It's clear that the Erin Brokovich star is in a good place right now.
"I feel super kind of happy," she said, when complimented by the magazine's guest interviewer Ryan Murphy on her appearance, explaining, "which I think translates to pretty, because my husband is home."