The present invention relates to a biosensor which comprises an electrode system including at least one pair of electrodes, at least one insulating base plate for supporting the electrode system, a first reaction layer provided at least on a working electrode of the electrode system, including an organic compound having a functional group capable of bonding or being adsorbed to an electrode and a hydrophobic hydrocarbon group, a second reaction layer provided on the first reaction layer, including an amphiphilic lipid capable of bonding or being adsorbed to a hydrophobic portion of the first reaction layer, and a reagent system carried in a two-component membrane composed of the first and second reaction layers, including at least membrane-binding type pyrroquinoline quinone-dependent glucose dehydrogenase and an electron mediator.