Knox was a prominent Edinburgh physician. He served as an army surgeon in Europe and South Africa, where he became interested in ethnological, zoological and medical research. On his return to Edinburgh in 1822, he was involved in establishing a museum of comparative anatomy and pathology at the College of Surgeons. At the same time, he built up a major anatomical school, probably the largest in Britain. By 1828 he had over 500 students.

Knox's reputation was severely damaged by his association with the murderers Burke and Hare - he had purchased 17 cadavers for dissection from them. Knox was not called upon to give evidence at their trial, and seemed surprised at the public anger directed towards him. Many found it hard to believe that such a prominent anatomist had failed to notice that the bodies he purchased had all undergone violent death, and had clearly not been recovered from graves. On 12 February 1829 an effigy of Knox was publicly hung before being torn to pieces in the street. He was vilified in popular ballads and caricatured in prints.

Despite being cleared of any knowledge of the murders by a medical committee, Knox was widely believed to have known more than he admitted about the origin of the cadavers he dissected and was progressively sidelined from the medical establishment in Edinburgh.

After failed attempts to establish a new anatomical practice, or gain a university position, Knox embarked on lecture tours, translated French anatomical textbooks and wrote for medical journals. While he was an opponent of slavery and critical of colonialism, Knox had strong views on racial difference. He was an early proponent of the race science which argued that humanity was divided into separate races, whose ability to acquire civilisation was very different.