The order of nanopores in anodic aluminium oxide (AAO) membrane formed by multi-step anodization in oxalic acid solution under a constant voltage of 40 V was studied. The average pore diameter increases slightly from 70 nm after two-step anodizing to 75 nm by six-step anodizing. The wall between neighboring pores becomes thinner from 40 nm after two-step anodization to 25 nm by six-step anodization. The interpore distance keeps a constant 105 nm. The orderly domain size is limited to micron size and increases for the two-step and three-step anodization, but the average size of the ordered domains remains constant after three-step anodization under the same working voltage. Moreover, surprisingly, “microflower-like” structures are observed over the surface of highly ordered AAO membrane after six-step anodic oxidation. We assume that they generate with the formation of the nanopores in the anodizing process.