A method for purifying bioactive substances includes the steps of: causing a bioactive substance having histidine units to contact media, each constituted by a substrate, ligands which are physically attached to the surface of the substrate, and Cu(II) or Fe(II) metal ions which are covalently bonded to the ligands; causing the bioactive substance to covalently bond with the metal ions via the histidine units; and washing the media with an amount of 1 nmol/L to 10 mmol/L imidazole derivative solution 60 times the volume of the media or greater. In the case that the metal ions are Cu(II), the bioactive substance which has covalently bonded with the Cu(II) via the histidine units are recovered by one of a 10 mmol/L to 1 mol/L imidazole derivative solution and a 0.5 mmol/L to 5 mol/L EDTA solution.