Born on 12 August 1910 in Trong, Taiping, Perak Darul Ridzuan, which was then part of the Federated Malay States, Yusof is the descendant of prominent Sumatran Malay Datuk Jannatun who migrated to Penang in 1759. Yusof is of Minangkabau descent from his father's side while his mother is a native Malay of Langkat region in Indonesia. His brother, Aziz Ishak, was a Malayan journalist and freedom fighter.

He received his primary education at Victoria Bridge School (now known as Victoria School), and secondary education at Raffles Institution. Yusof was well known both as a journalist and the founder of the Malay newspaper Utusan Melayu prior to becoming head of state of Singapore. He was married to Noor Aishah.

He first served as Yang di-Pertuan Negara (head of state) between 1959 and 1965, remaining in office during the time that Singapore was part of the Federation of Malaysia between 1963 and 1965. Following Singapore's departure from Malaysia in 1965, he served as the first President of the Republic until his death in 1970.

Yusof is buried at Kranji State Cemetery.