The recording will play at the exhibition at the great gatehouse of Battle Abbey in East Sussex which was built on the apparent spot where English King Harold was killed by William the Conqueror in 1066.
The carol, called Be Mery, was found scribbled beside a short poem and remedies for stomach problems at the back of a monk's service book dating from around 1500.
It will be the first time the song has been heard since the Reformation.
During the Medieval era, carols were sung all year round and would have been sung and danced to at social occasions as well as during church services.
The long-lost carol was found by Dr Michael Carter at the library of Trinity College, Cambridge.
Dr Carter said: "This carol is clear evidence that the Battle monks were very much part of the thriving devotional culture of Catholic England, a culture brutally cut short by the Reformation and the dissolution of the monasteries which extinguished 500 years of religious life at Battle.
"Our new exhibition has been designed to reveal insights into that life, the importance of prayer and worship, the tremendous power and wealth of the abbey and what it was like to be an ordinary monk there.
"The carol is very exciting and, by setting it to music for the first time in 500 years, we hope to give visitors to the abbey a real insight into the lives of the monks who lived at the abbey."
The new recording has been performed by chola Gregoriana: The Association for Gregorian Chants.
The new exhibition at the great gatehouse will open to the public on Saturday.