A new fluorescence enhancement technical platform based on anodic aluminum oxide (AAO) nanostructure substrate is reported for the first time. Several fluorophores have been examined on the AAO nanostructure substrates. Systematic experiments found that the enhancement factor can be up to two orders of magnitude compared to the fluorescence signals on a glass substrate, indicating its great potential for ultrasensitive fluorescence detection. Given the simple and cost-effective fabrication process of lithographically patterned AAO nanostructure, this type of AAO nanostructure platform has great potential applications, especially its integration with microdevices and microfluidic devices for fluorescence-based biological analysis.