An object of the present invention is to provide a biosensor that can detect binding of a compound with a functional protein and then assay a reaction product derived from the activity of the functional protein again. The present invention provides a biosensor for detecting a test molecule specifically binding to a physiologically active substance, which comprises; (1) (a) a first reaction region on which the physiologically active substance has been immobilized for performing a binding reaction between the physiologically active substance and the test molecule and a physiologically active reaction caused by the physiologically active substance, and (b) a second reaction region on which a molecule that specifically binds to a reaction product resulting from the physiologically active reaction has been immobilized for performing a binding reaction between the reaction product and the molecule that specifically binds to the reaction product, in the same area; and (2) an assay region for detecting changes in the binding reaction in the first reaction region and in the binding reaction in the second reaction region.