The fluorescence emission and enhancement of Rhodamine 6G on the silver island films (SiFs) with and without 4-Aminothiophenol molecules have been investigated. It was found that the SiFs without 4-Aminothiophenol molecules (PATP) have less contribution to the fluorescence enhancement. However, when PATP molecules as a spacer were deposited on the SiFs, obvious fluorescence enhanced effect was observed. The results show that the fluorescence enhancement depends on the morphology of the substrate surface and the distance between fluorophore molecules and the substrate surface. The experimental observations can be explained by local surface plasmon resonance and local field enhancement theory.