This paper describes a glucose biosensor based on glucose oxidase (GOx) immobilized on porous ZnO/Au electrodes. The ZnO porous electrodes were fabricated by electrochemical deposition of zinc oxide on patterned Au electrodes using polystyrene (PS) spheres as templates. Uniform pore size and highly ordered ZnO pore arrangement were observed from SEM images. X-ray diffraction (XRD) patterns revealed a single crystalline nature of the porous ZnO. This porous structure provides high enzyme loading capacity and long-term stability. In a pH 7.4 phosphate buffer solution, the positively charged high isoelectric point (IEP) ZnO pores enhance the adsorption of negatively charged low IEP GOx through electrostatic attractive force. Once GOx molecules are immobilized within the ZnO pores, the bottleneck structure resulting from the connected pores hinders leaching of GOx from the pores. The resulting enzymatic biosensor showed a linear detection range from 1mM to 18mM, and sensitivity of 10.89μA/ (mM·cm2) with good selectivity and long-term stability.