Born in Cambridge, Osbert was the only child of biologist John Edmund Sharrock Moore and Heloise Moore (née Salvin). He was named after Helloise's father, the naturalist Osbert Salvin. He studied modern languages at Exeter College, Oxford. He helped a friend to run an antiques shop before joining the army at the outbreak of World War II, joining the anti-aircraft regiment before being transferred to the Intelligence Corps officer-cadet training camp. He was posted to a camp on the Isle of Man to help oversee Italian internees.
In 1944 he was posted to Italy serving as an intelligence officer interrogating spies and saboteurs. During this period he discovered Buddhism via Julius Evola's The Doctrine of Awakening a Nietzschean interpretation of Buddhism. This work had been translated by his friend Harold Edward Musson, also an intelligence officer serving in Italy.
After the war Moore joined the Italian section of the BBC. Moore and Musson, who shared a flat in London, were quite disillusioned with their lives and left to Sri Lanka in 1949 to become Buddhist monks. On 24 April 1949 they received the novice (samanera) ordination or going forth, pabbajjā, from Ñāṇatiloka at the Island Hermitage. In 1950 they received their bhikkhu ordination at Vajirarama Temple Colombo. Ñāṇamoli spent almost his entire monk life of eleven years at the Island Hermitage.
After having been taught the basics of Pali by Nyanatiloka Mahathera, Ñāṇamoli acquired a remarkable command of the Pali language and a wide knowledge of the canonical scriptures within a comparatively short time. He is remembered for his reliable translations from the Pali into English, mostly of abstruse texts such as the Nettippakaraṇa which are considered difficult to translate. He also wrote essays on aspects of Buddhism. By 1956 he had translated Visuddhimagga into English and got it published as The Path of Purification. He also compiled The Life of the Buddha, a reliable and popular biography of the Buddha based on authentic records in the Pali Canon. His notes with his philosophical thoughts were compiled by Nyanaponika Thera and published as A Thinker's Note Book.
His handwritten draft translation of the Majjhima Nikaya was typed out after his death and edited by Bhikkhu Khantipalo, and partly published as A Treasury of the Buddha's Discourses and then edited again by Bhikkhu Bodhi and published as Middle Length Length Discourse of the Buddha and published by Wisdom Publications in 1995. Other draft translations, edited and published after his death, are The Path of Discrimination (Paṭisambhidāmagga) and Dispeller of Delusion (Sammohavinodanī).
While on a pilgrimage he died suddenly due to heart failure at the hamlet of Veheragama near Mahawa. His body was brought to Vajirarama Temple in Colombo and cremated at a nearby cemetery.