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The fabrication of a ZnO/Au nanosquare-array electrode was successfully carried out for the detection of glucose concentration in biomedical applications. The fabrication of the ZnO/Au nanosquare array using an ultra-thin alumina mask (UTAM) based on the imprinted anodic aluminum oxide (AAO) template and the direct current (DC) sputtering method was able to produce a very well-ordered nanosquare arrangement with a side size of 300 nm and a thickness of 100 nm. Tests were done to evaluate the performance of the electrode by means of cyclic voltammetry (CV) which showed that the addition of glucose oxidase (GOx) increased the sensitivity of the electrode up to 1180 ± 116 μA mM−1cm−2, compared with its sensitivity prior to the addition of GOx of 188.34 ± 18.70 mA mM−1 cm−2. A iox/ired ratio equal to ~1 between the peaks of redox reactions was obtained for high (hyperglycemia), normal, and low (hypoglycemia) levels of glucose. The ZnO/Au nanosquare-array electrode was 7.54% more sensitive than the ZnO/Au thin-film electrode. Furthermore, finite-difference time-domain (FDTD) simulations and theoretical calculations of the energy density of the electric and magnetic fields produced by the ZnO/Au electrode were carried out and compared to the results of CV. From the results of CV, FDTD simulation, and theoretical calculations, it was confirmed that the ZnO/Au nanosquare array possessed a significant optical absorption and that the quantum effect from the nanosquare array resulted in a higher sensitivity than the thin film.