Abstract: Using the principle that the fluorescence of butyl rose red B (BRMB) can be quickly quenched by H₂O₂-Fe²⁺, and catalase (CAT) can inhibit this quench process, a method for the determination of activity of serum CAT by fluorescence quenching method based on H₂O₂-BRMB-Fe²⁺ system was proposed. The serum sample (10 μL) was taken, and 0.50 mL of 1 mmol·L^-1 H₂O₂ base solution was added, and the reaction was performed at 30 ℃ for 15 min. Then 0.70 mL of 0.02 mmol·L^-1 BRMB solution, 1.00 mL of 0.1 mol·L^-1 glacial acetic acid-sodium acetate buffer solution (pH 4.2) and 0.08 mL of 0.01 mol·L^-1 Fe²⁺ solution were added successively, and the mixed solution was reacted at room temperature for 20 min. After its volume was made up to 10 mL with water, the fluorescence intensity F of system was measured at the emission wavelength of 590 nm. The boiled dead enzyme solution was used as control, and the fluorescence intensity F₀ was measured at the emission wavelength of 590 nm using the same method. It was shown that linear relationship between CAT activity and fluorescence intensity difference (F-F₀) was kept in the range of 0.005-0.05 U·mL^-1, with detection limit (3s/k) of 1.2×10^-3 U·mL^-1. The method was applied to analysis of activities of 6 serum CAT from healthy people of different ages, and the determined values were in the range of 31.50-72.30 U·mL^-1, which were basically consistent with iodimetry. The spiked recoveries of CAT activity were in the range of 95.2%-102%, and RSDs (n=6) of the determined values ranged from 1.7% to 3.3%.