The trainer had warned time could be called on the 10-year-old's career should he fail to fire in the Unibet International Hurdle and after being pulled up before the last, Twiston-Davies confirmed The New One will not race again.
The New One bows out the winner of two Grade Ones and over £1million in prize money for the Twiston-Davies team.
The trainer said: "It is very emotional. It was great to see him go out there like that, enjoying himself. Sam (Twiston-Davies, jockey) said when push come to shove, he doesn't have it in him anymore.
"I'm absolutely gutted as where can I find another one like him from? I really hoped he would come back today. There are absolutely no excuses."
The New One has been a stalwart on the top-level hurdling scene since claiming a first Grade One in the two-mile-five-furlong novices' hurdle at the 2013 Cheltenham Festival.
He was unfortunate not to win the Champion Hurdle the following season when suffering a troubled passage, but made amends when adding another Grade One win in the Aintree Hurdle.
The New One also counts three International Hurdle successes on his record of 20 wins in 40 starts, as well as notching a string of places in the highest company in the hands of the trainer's son.
Twiston-Davies added: "There have been lots of special moments and lots of disappointments as he should have won the Champion Hurdle but he didn't.
"He was a great help getting Sam going. He has been the most wonderful horse.
"He has been part of the family. He is not going away anywhere and we will look after him."
