Johnstone qualified and practised as a solicitor in Edinburgh but he decided to abandon law for art. He studied art at evening classes before visiting Italy between 1842 and 1844. Johnstone was elected an associate of the Royal Scottish Academy in 1840 and a full academician in 1848, and contributed work each year to the annual exhibitions. He became treasurer to the academy in 1850 and acted as librarian between 1853 and 1857. Johnstone was involved in the establishment of the National Gallery of Scotland that opened in 1859 and became the Gallery's first principal curator and keeper, greatly enriching the collection. He continued to work for both the National Gallery and the academy until his death.