An electrochemical biosensor includes a substrate, a plurality of layered active metal parts, a plurality of layered electrodes, a reaction confinement layer, an electrochemical reactive layer and a cover piece. The substrate is formed with through holes each of which is defined by an interior wall surface and penetrates top and bottom surfaces. Each of the layered active metal parts is formed at least upon a respective one of the interior wall surfaces. The layered electrodes are formed on the layered active metal parts. The reaction confinement layer confines a reactor space over a region where the through holes are formed. The electrochemical reactive layer is disposed in the reactor space and is electrically coupled to the layered electrodes.