In this work, the silver nanoparticle (Ag NP) films were deposited on glass slide by dc magnetron sputtering. The effect of annealing temperature on morphology and optical property were characterized by field-emission scanning electron microscopy (FE-SEM), atomic force microscopy (AFM) and UV–Vis-NIR spectroscopy, respectively. From the results, the size and inter-particle distance of Ag NPs depend on annealing temperature. The localized surface plasmon resonance (LSPR) band position shift to the blue with the annealing temperature increased. The surface enhanced fluorescence (SEF) effect of the Ag NPs films was investigated by emission spectra of Rhodamine 6G (Rh6G) fluorescent molecules. It was found that the florescent intensity of R6G, corresponding to the diameter and surface roughness of Au NP films. Finally, the Ag NPs film substrate offer a cost-effective with non-lithography fabrication potentially applied in plasmonic sensor chips for biomedical and food safety applications.