Abstract: Fluorescein isothiocyanate (FITC) can emit strong and stable fluorescence，KIO₃ can oxidize FITC and quench the fluorescence signal，Se(Ⅳ) can strongly quench the fluorescence signal of FITC, and the content of Se(Ⅳ) is directly proportional to ΔF. Based on the facts above，a new fluorescence quenching method for the determination of trace Se(Ⅳ) has been established based on the catalytic oxidiation of FITC by KIO₃. The linear range of this method was 0.012~2.000×10^－15 g Se(Ⅳ)/mL，with a detection limit of 2.1×10－18 g/mL, n=11，the regression equation of working curve was ΔF=1.18＋72.74C_Se(Ⅳ)(×10^－15 g/mL)，n=6，r=0.9997. Samples containing 0.012 and 2.000×10^－15 g Se(Ⅳ)/mL respectively were measured for seven times and the RSDs were 2.8% and 3.5%. It has been satisfactorly applied to the determination of trace Se in real samples and has used to diagnose and forecast human disease. The mechanism for the determination of trace Se(Ⅳ) by catalytic fluorimetry was also discussed.