Viewers were left stunned when the talking dog died in the latest episode.
The character, voiced by Family Guy creator Seth MacFarlane, was killed off in an episode called Life of Brian after he was hit by a car.
The fans have signed a petition on Change.org to resurrect Brian Griffin.
Brian Griffin, a family dog that talks, drinks, goes out with women and is an aspiring novelist, has been a major character since the pilot of Family Guy in 1999.
The character has appeared in more than 200 episodes of the animated cartoon comedy.
Family Guy, which is set around bumbling father Peter Griffin, his wife Lois, teenage children Chris and Meg and baby Stewie, is known for its raunchy humor.
The show has been criticized by watchdog groups in the US and episodes often feature characters in compromising or inappropriate situations.
The Emmy-nominated series is in its 12th season and averages 6 million viewers an episode in the US.
In Sunday’s episode, the Griffin family is left distraught as Brian dies saying: “You’ve given me a wonderful life. I love you all.”
The family holds a funeral and shares memories of their friend, with Peter Griffin saying: “Brian wasn’t just my dog, he was my best friend in the whole world.”
Later in the sixth episode of Season 12, the family adopts a new dog, smart-talking Vinny voiced by Sopranos actor Tony Sirico, who quickly becomes part of the family even winning over Stewie by the end of the show.
Representatives for Fox have so far declined to comment on Brian Griffin’s death.
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