Abstract: A series of fluorescent monodisperse polystyrene microspheres labeled with 1, 8naphthamide dyes were prepared by dispersion copolymerization. Making use of fluorescence resonance energy transfer (FRET), the prepared microspheres showed both the fluorescence of the donor and acceptor dyes under the excitation wavelength of the donor dye. It is found that effective FRET occurs at a relatively low dye concentration and appropriate molar ratio of donor and acceptor. An increase in the dye concentration, which reduces the distance between the donor and acceptor, leads to more effective FRET in microspheres. By changing the dye concentrations, microspheres could show tuned fluorescent signals under a single wavelength excitation. This type of uniform fluorescent microspheres has potential application in bioassays.