Fans of the long-running PBS children's show recently began an online campaign to get the puppets to tie the knot, following the legalisation of gay marriage in New York.
The petition read: "We are not asking that Sesame Street do anything crude or disrespectful by allowing Bert and Ernie to marry. It can be done in a tasteful way.
"Let us teach tolerance of those that are different. Let Sesame Street and PBS Kids be a big part in saving many worthy lives."
However, Sesame Workshop - the non-profit organisation behind Sesame Street - has now issued a statement claiming that Bert and Ernie do not have any "sexual orientation" and are simply "best friends".
"Bert and Ernie are best friends," the statement read. "They were created to teach preschoolers that people can be good friends with those who are very different from themselves.
"Even though they are identified as male characters and possess many human traits and characteristics (as most Sesame Street Muppets do), they remain puppets, and do not have a sexual orientation."
Bert and Ernie's relationship has been the subject of much speculation over the course of the programme's 42-year history.
Last year, Bert posted on the show's official Twitter feed: "Ever notice how similar my hair is to Mr T's? The only difference is mine is a little more 'mo', a little less 'hawk'." Some fans took this as a covert reference to Bert being gay, since "mo" is a slang term for homosexual.
Lady GaGa, Neil Patrick Harris and Ellen Degeneres were among the celebrities who celebrated the legalisation of gay marriage in New York earlier this year.
Watch a clip of Bert and Ernie on Sesame Street below: