Highly fluorescent silver nanoparticles (AgFNPs) have been prepared by microemulsion method and the sizes of AgFNPs were controlled by altering the molar ratio (ω) of water-to-surfactant in the water-in-oil microemulsion. The results were shown that the AgFNPs sizes increased with incremental molar ratio (ω) of water-to-surfactant. The AgFNPs have been characterized by transmission electron microscopy, dynamic light scattering, fluorescence and absorption spectroscopy, and fluorescence lifetime study. Study of the spectral characteristics was shown that the absorbance of AgFNPs increased significantly with the ω, and linear relationship between absorbance and the size of AgFNPs was observed. The increase of AgFNPs size caused a red shift of maximum absorption wavelength in the UV–Vis spectra, and the relationship between maximum absorption wavelength and AgFNPs size appeared linear dependence. The maximum fluorescence emission wavelength did not shift with the change of particles size, but the emission intensity increases with the ω. The results were shown that the other factors to affect the fluorescence properties of AgFNPs were the surface properties and microstructure, except the AgFNPs size. These surface properties depend upon the stabilizing agent, reactant concentration, and solvents and so on.