Northern rockhopper Mrs Wolowitz hatched in 1987 and this year celebrated her 35th birthday marking double her life expectancy.
The flightless bird - named after a character in the TV sitcom The Big Bang Theory - was killed by a fox that managed to enter her enclosure in the early hours of Thursday morning.
Edinburgh Zoo is famed for its penguins, which also include king penguins and gentoos, and keepers had described Mrs Wolowitz as "the oldest and sassiest penguin in the colony".
No other penguins were harmed in the attack, which has saddened keepers and the penguin's many fans alike.
Darren McGarry, head of living collections at the Royal Zoological Society of Scotland (RZSS) said: "We were saddened to discover a fox had broken into our penguin enclosure through the night and attacked Mrs Wolowitz, our oldest penguin.
"She was a big personality and a favourite among keepers and visitors, who will miss her. Thankfully, the rest of our colony were unharmed and are doing well.
“Our expert teams check the enclosures daily for signs of damage but there is always a risk that wild animals will break in. We will be looking at what we can do to reinforce the boundary."
Rockhoppers - among the smallest penguins and known for their bright yellow head plumage and red eyes - usually live around ten years in the wild and around twice that age in captivity.
Mrs Wolowitz celebrated her 35th birthday on May 13, when she was presented with a birthday treat of her favourite sprats and whitebait by keepers at the attraction's famous Penguins Rock, home to Europe's largest outdoor penguin pool.
Dawn Nicoll, senior animal keeper at Edinburgh Zoo, said at the time that Mrs Wolowitz was "extraordinary".
Speaking on the penguin's birthday, she added: "Mrs Wolowitz hatched at Edinburgh Zoo so she's always been with us and in fact she's older than most of her keepers.
"We all have a soft spot for Mrs Wolowitz. She always gives us a honk when we arrive in the morning and then when we shout 'goodnight' at the end of the day.
"She also has a bit of a fan club among our visitors, who look out for her in her favourite sunny spot. But she can be quite feisty. She'll stand her ground with the juvenile penguins and isn’t afraid to tell them off if they are in her space or annoying her."
Mrs Wolowitz produced her only offspring, Mr Green, in 1991, when she was four years old.
The pair were inseparable and Mrs Wolowitz was named at that time after a mother character in the TV sitcom The Big Bang Theory, dedicated to her son, Howard. Mr Green reached the age of 25 before he passed away in 2016.
Penguin fans posted messages of condolence online. One, Louise Wilson, wrote: "Such a shame poor wee thing, sad for the keepers and her other penguin family."