Biosensor and method for quantitative measurement of a substrate using the same

A biosensor is disclosed for application to a method for quantitative measurement of a substrate comprising a first step of causing a substrate contained in a sample to react with an oxidoreductase specific to the substrate in the presence of an electron mediator in oxidized state, a second step of applying a potential to a working electrode for reducing the electron mediator in oxidized state that remains not reduced in the first step, and a third step of measuring a reduction current flowing across the working electrode and a counter electrode. The biosensor comprises an electrically insulating base plate, an electrode system having a working electrode and a counter electrode formed on the base plate, and a reaction layer which is formed on or in the vicinity of the electrode system and contains at least an oxidoreductase and an electron mediator, the counter electrode including at least a reductant of a redox compound or an electrolytically oxidizable metal.