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