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Mesoporous aluminum oxide (MAO) films with perpendicularly oriented cylindrical mesopores (pore diameter: ca. 10 nm) were successfully deposited on a glass substrate by a surfactant-templating approach using aluminum nitrate as an aluminum source. The perpendicular orientation of mesopores was confirmed by grazing-incidence small-angle X-ray scattering and neutron reflection experiments. The thickness of the MAO film was around 100 nm, with a surface roughness of less than 6 nm. Since the inner surface of MAO pores was positively charged, negatively charged glucose oxidase molecules could be densely loaded into the cylindrical mesopores without significant loss of enzymatic activity. The present MAO film is potentially useful as an inorganic host material for an enzyme toward the development of a biocatalytic system.