It is an object of the present invention to provide a biosensor wherein a reference unit and a measurement unit can be prepared by performing only a single operation for immobilizing a physiologically active substance and wherein unnecessary electric charge does not remain in the reference unit. The present invention provides a biosensor which comprises a substrate composed of a metal surface or metal film coated with a hydrophilic polymer compound, and which has a surface for retaining a physiologically active substance and a surface that does not retain a physiologically active substance on a single plane of the substrate.