Nottinghamshire's Steve Ward, who was first recognised as the oldest fighter in 2011, lost his crown to an American earlier this year.
But he regained the title in a fight against Jody Meikle, 35, which he lost in the third round.
Ward said after the bout: "The best guy won on the day - he was too strong for me, but at least I got the record."
The boxer, who does not plan to retire yet, said "age was a factor" for the first time in his career.
"I got put down a few times, but I kept getting up," he said.
Ward, who grew up in Nottingham and now lives in Mansfield and works in Derbyshire, turned professional in 1977 and retired in 1987 before making a comeback in 2010.
He said: "I am alright - a slight bruise on my left ear, but what really hurts me more than anything is my left arm."
His promoter, former boxer and gym owner Roger Brotherhood, said: "I think he's fantastic and he is credit to his age.
"He is an inspiration and his preparation was second to none. I can't fault him... he has looked after himself inside and outside the ring."
Ward added: "I always fight younger guys, but now I am going to go for boxers who are closer to my age."
The previous Guinness Book of Records oldest boxer was American Mike Palmer, who was almost 58 when he fought in North Dakota in February.
A British cruiserweight has regained his bragging rights as the world's oldest professional boxer at 59.