The invention discloses an affinity chromatography column based on immobilized thrombin, and a preparation method and an application thereof, and belongs to the fields of chromatographic science, biology and the like. Glutaraldehyde is bonded to an aminopropyl silica gel, and a thrombin solution and the glutaraldehyde form covalent bonds to make the thrombin immobilized on the surface of the aminopropyl silica gel in order to prepare a thrombin stationary phase; and the thrombin stationary phase is loaded into a column through a wet process to prepare the bio-affinity column based on immobilized thrombin. The affinity column has a good stability and a high specificity, can be used for screening direct thrombin inhibitors, and lays a foundation for the commercialization of bio-affinity chromatography columns.