A biosensor is provided. The biosensor includes a lower substrate including an electrode unit, an insulation layer disposed on the lower substrate, a first spacer layer disposed on the insulation layer over the electrode unit, an enzyme unit disposed on the first spacer layer, a second spacer layer disposed on the enzyme unit, such that the enzyme unit is interposed between the first and second spacer layers, and an upper substrate disposed on the second spacer layer. The electrode unit includes a working electrode, and a reference electrode and a counter electrode that surround a periphery of the working electrode, facing the working electrode.