An object of the present invention is to provide a method for production of a physiologically active substance-immobilized substrate, which can prevent the inactivation of a physiologically active substance and/or can immobilize a physiologically active substance at a high concentration onto a substrate surface without use of electrostatic attraction. The present invention provides a method for production of a physiologically active substance-immobilized substrate, which comprises steps of: applying, to a metal substrate having a layer for immobilizing a physiologically active substance, a solution containing a physiologically active substance capable of forming a covalent bond with a molecule constituting the layer for immobilizing a physiologically active substance; and then drying the solution.