We investigated an electrochemical separation method for anodic aluminum oxide (AAO) films possessing thicker barrier layers by electrolysis in sodium chloride (NaCl) and ethylene glycol (EG) solution and subsequent fabrication of through-hole AAO membranes by immersion in phosphoric acid (H3PO4) solution. The AAO films with different barrier layer thicknesses were formed on the aluminum surface by anodizing in sulfuric, oxalic, and H3PO4 solutions at up to 130 V. The AAO-covered aluminum specimens were immersed in NaCl/EG solution, and then constant voltage electrolysis at 10 V higher than the anodizing voltage was performed for the separation of AAO from the aluminum surface. Many nanoscale paths were formed throughout the barrier alumina layer in the initial stage of electrolysis, and then the aluminum substrate electrochemically dissolved through these narrow paths during electrolysis. Although the AAO film formed by anodizing at 130 V was partially fractured by electrolysis, the AAO films formed at up to 80 V were uniformly separated from the aluminum surface. As the separated AAO membrane was immersed in H3PO4 aqueous solution after electrolysis, the barrier layer with narrow paths preferentially dissolved into the solution, and a through-hole AAO membrane could be successfully obtained.