Molecularly imprinted polymers (MIPs) were prepared by a one-step swelling polymerization method in aqueous system using nano-polystyrene microspheres which was gained by dispersion polymerization as seeds and using Rhodamine B as template molecules. The infrared spectrometry(IR) of the polymers further indicated that there were some functional groups in the MIPs which could interact with the template. The binding mechanism and identification characteristics of MIPs were evaluated by ultraviolet spectrometry. Morphological characterizations of the MIPs were tested by scanning electron microscope (SEM); The adsorption ability and fluorescence characteristics were evaluated by fluorescence spectrophotometry. Results show that the diameters of PS microspheres were between 100 〜150nm and the diameters of MIPs were between 200 〜300nm. Rhodamine B molecularly imprinted polymer of Rhodamine B molecules with specific recognition selectivity and excellent combination of properties The maximum excitation wavelength and the maximum emission wavelength were 557 nm and 593 nm.