Micropatterns of the nanostructured aluminum oxide (NAO) with sizes from 5 to 200 mu m have been successfully fabricated on the indium tin oxide (ITO) glass substrate by simply combining a lift-off process and a one-step anodization process for the first time. The detection of fluorescent dyes and biomolecules tagged with fluorescent dyes on the NAO has been investigated and demonstrated successfully. Experiments reveal that the micropatterned NAO substrates can increase the fluorescence signals up to 2 or 3 orders of magnitude compared to the glass substrate, suggesting a possibility to significantly reduce the consumption of the biosamples for fluorescence-based sensing, imaging, and analysis. The stability of the NAO substrates for fluorescence enhancement has also been evaluated by monitoring the fluorescence signals after the fluorophores applied on the substrates for a period of time and reusing the same NAO substrates many times. It was found that this type of substrate has very good stability. Because the micropatterned NAO can be easily integrated with microsensors or microfluidic chips, a simple and inexpensive fluorescence enhancement platform can be developed for a variety of applications, such as microarray technology and single-cell imaging, facilitating the construction of the on-chip fluorescence-based micro- or nanosystems.