Three-dimensional (3D) plasmonic nanostructures of porous alumina array (PAA) with silver nanoparticles (AgNPs) were prepared for enhancing fluorescence emission. In order to avoid fluorescence quenching effects and obtain clear fluorescence enhancement, the molecules were separated by using such 3D substrates, and the mean distance between the molecules and nanoparticles' surface can be easily controlled by changing the diameters of the PAA tube. It was found that the PAA tube with smaller size provides better fluorescence enhancement. Enhanced cross section, a new fluorescence enhanced factor, combined with the simulation of localized electromagnetic field enhancement was presented to understand the experimental results.