John Macoun was born on April 17, 1831, in Magheralin, County Down, Northern Ireland.

He was a naturalist. In 1850, his family emigrated to Ontario, Canada, and began farming. In 1856, unsatisfied as a farmer, he became a school teacher and developed a deep interest in botany. His knowledge and dedication to fieldwork became sufficiently advanced and in 1868, he was offered the position of Professor of Botany and Geology at Albert College in Belleville. Between 1872 and 1881, he participated in five surveying expeditions to the Pacific and the Northwest for the proposed Canadian Pacific Railway. A major purpose of these expeditions was to determine the agricultural potential of various regions of the west. In 1879, the Geological Survey of Canada appointed him Explorer of the Northwest territories and moved his family to Ottawa where he served as Botanist to the Geological and Natural History Survey of Canada for the next 31 years. In 1887, he became its Assistant Director and in 1882, a charter member of the Royal Society of Canada. He was a prolific collector and cataloguer of Canadian flora and fauna. Over 100,000 samples from his collection of plants are housed in the National Herbarium of Canada, Canadian Museum of Nature, Ottawa.

In 1862, he married Ellen D. Terrill (1841–1922). He died on June 18, 1920, in Sidney, British Columbia.