It was found that the flourescence intensity of rufloxacin hydrochloride was enhanced in the precense of CTMAB and β CD. A simple and highly sensitive spectrofluorimetric method was proposed. At the range of rufloxacin hydrochloxide of 6.58×10 -8 ~5.0×10 -6 mol/L, the value of the fluorescence intensity appeared good linear relations with the concentrations of RFLX. The relative coefficient was 0.9998 and the RSD was 0.9%. The detective limit was 2.19×10 -9 mol/L.