It is an object of the present invention to provide a biosensor, wherein non-specific adsorption on a surface that does not retain a physiologically active substance (reference unit) is suppressed. The present invention provides a biosensor which comprises a substrate having at least two types of surfaces on a single plane, wherein at least one of said surfaces does not retain a physiologically active substance, and wherein at least two types of hydrophilic compounds with different molecular weights are bound to said surface that does not retain a physiologically active substance.