With silicon substrates and arrays of optical fiber cores as assembly fixtures, 3×3 arrays of Au microspheres produced by melting and condensing Au fibers were manually arranged. ZnO nanowires were hydrothermally synthesized on Au microspheres, and glucose oxidase was immobilized on ZnO nanowires, obtaining the spherically hierarchical structure array-based working electrodes. The surface morphologies and the glucose oxidase immobilization effects of the spherically hierarchical structures as well as the electrochemical performance of the working electrodes were quantitatively characterized. The linear range, sensitivity, low detection limit and Michaelis-Menten constant of this glucose sensor are 0~2.5 mmol/L, 17.24 μA·(mmol/L)-1·cm-2, 3.44 μmol/L, and 1.85 mmol/L respectively.