A novel method was established for the rapid and nondestructive determination of the barrier layer thickness of anodic aluminum oxide (AAO) films based on their optical transmission spectra. Nanoporous AAO films with highly ordered pores have been prepared by a two-step anodization approach. Series UV-VIS-NIR transmission spectra of AAO with different barrier layer thickness have been studied. Oscillatory waves were observed at visible-near infrared region in the spectrum of AAO with the existence of barrier layer. Furthermore, a linear relationship between the number of wave crests and the thickness of barrier layer was established, for which the correlation coefficient is 0.99979. This relationship affords a simple and efficient method for rapid and nondestructive detection of the barrier layer thickness of AAO films.