[Objective] To investigate the size-controlled preparation of highly ordered anodic aluminum oxide (AAO) membranes, which have a wide range of brand-new applications in the nanotechnology and biotechnology. [Methods] The AAO membranes were prepared following the two-step anodization process. And the orthogonal tests were used to synthetically study the influences of the electrochemical parameters on the micro-performances of AAO membranes. [Results] From the results of orthogonal tests, the integrated impact of the anodization parameters, including the applied potential, electrolyte, temperature and time, on the average pore diameters, thickness and regularity of the AAO membranes were concluded. The average pore diameters were mainly affected by potential and electrolyte. While, the thickness and regularity were dependent on the cooperation of the temperature, potential and electrolyte, and the current density can indicate the integrated impact of those parameters. And a series of highly ordered AAO membranes, with defined size were also prepared. [Conclusions] The strategy of preparing highly ordered AAO membranes with defined pore diameter and thickness, in the mild condition, was obtained. A series of highly ordered AAO membranes, with pore diameter between 10~70 nm in 15~ 25 ℃ have been successfully prepared in this way.