There is disclosed a method for producing a molecule immobilizing substrate, comprising at least the steps of: forming, on a substrate, a monomolecular film including hydroxyl groups, cyano groups, or oxiranyl groups, which are oriented toward an outmost surface of the monomolecular film; and chemically modifying the hydroxyl groups, cyano groups, or oxiranyl groups of the monomolecular film to transform them into carboxyl groups, to thereby form, on the substrate, the monomolecular film including the carboxyl groups, which are oriented toward an outmost surface of the monomolecular film. There can be provided: a method for producing a molecule immobilizing substrate which is free of occurrence of an immobilized-molecule peeling problem in the case of conducting an assay by immobilizing molecules on the substrate.