Mesoporous zirconia thin films (MZFs) were synthesized using zirconium hydroxide sol particles and a structure directing agent, Pluronic F127 (PEO106PPO70PEO106, EO = ethylene oxide, PO = propylene oxide). By controlling the F127/Zr ratio, we obtained two distinct MZFs with one in the Fmmm structure and the other in the P63/mmc structure. The pore structures of these films were characterized by low-angle X-ray diffraction, grazing incidence small-angle X-ray scattering, electron microscopy, and N2 sorption measurement. The Fmmm structure has interconnected pores and the P63/mmc structure has less accessible pores. The MZFs were functionalized with glucose oxidase (GOx) and were studied for their potentials as an electrochemical sensor for glucose. The GOx-functionalized MZF electrodes show high sensitivity to glucose in a broad range of glucose concentration of 0.025 - 6.8 mM, which can be attributed to their biocompatibility providing a favorable microenvironment for GOx immobilization and to their 3D pore structures with good accessibility of pores.