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The fabrication of nanopore membrane by deposition of Al2O3 film using electron-beam evaporation, which is fast, cost-effective, and negligible dependency on substance material, is investigated for potential applications in water purification and sensors. The decreased nanopore diameter owing to increased wall thickness is observed when Al2O3 film is deposited on anodic aluminum oxide membrane at higher deposition rate, although the evaporation process is generally known to induce a directional film deposition leading to the negligible change of pore diameter and wall thickness. This behavior can be attributed to the collision of evaporated Al2O3 particles by the decreased mean free path at higher deposition rate condition, resulting in the accumulation of Al2O3 materials on both the surface and the edge of the wall. The reduction of nanopore diameter by Al2O3 film deposition can be applied to the nanopore membrane fabrication with sub-100 nm pore diameter.