The fluorescence enhancement effect of Rh6G molecules deposited on the silver film substrate decorated with nanohole arrays was investigated in this paper. The prepared substrate, decorated with nanohole arrays, was fabricated with the deposition of silver films onto the anodic aluminum oxide templates through magnetron sputtering method. Compared with the conventional continuous silver film substrate, the prepared substrate shows better enhanced effect. Particularly, the fluorescence enhancement factor has a relationship with the size and period of the nanohole arrays. The experimental observations were analyzed with local surface plasmon resonance model. The results of current work highlight the importance of strong electromagnetic coupling effect in surface enhanced fluorescence.