The Griffins' much-loved dog died when his best friend Stewie failed to revive him with his time machine after he was hit by a car.
The change.org online petition states: "The writers of Family Guy didn't just kill off one of their creations, they killed off the dog who has lived in our homes for the last 15 years. They killed the dog we all have come to love. They killed America's dog!"
The petition paid tribute to the "witty and sophisticated element" that Brian had brought to the show and described him as "an important part of our viewing experience".
"Family Guy and Fox Broadcasting will lose viewers if Brian Griffiin is not brought back," avid viewer Aaron Thompson wrote.
Fans have also been 'liking' the 'R.I.P Brian Griffin from Family Guy' page on Facebook. Close to 750,000 people have joined with the most dedicated supporters creating tribute art and memes in the character's honour.
Creator Seth MacFarlane and the Family Guy production team introduced new dog Vinny towards the end of the show in a move that served only to anger fans more.
Producers are confident Family Guy fans will move on from Brian's death and warm to Vinny, who is voiced by Tony Sirico from The Sopranos.
"Our fans are smart enough and have been loyal to our show for long enough to know that they can trust us," said Callaghan. "We always make choices that work to the greatest benefit of the series."