Abstract: Fluorescence with definite intensity was emitted by rhodamine B with λ_ex of 365 nm and λ_em of 580 nm in a reaction system containing 2.0 mL of 0.1 mol·L^-1 H₂SO₄ solution, 2.0 mL of 0.1 mol·L^-1 KI solution and 5.0 mL of 1.0×10^-5mol·L^-1 rhodamine B solution, with total volume of the reaction solution of 50.0 mL. When peroxy acetic acid was added to the reaction system, the fluorescence was quenched instantly. It was found that linear relationship was kept between the magnitude of decrease of fluorescence and concentration of peroxy acetic acid in the range from 4.2×10^-7 to 5.0×10^-5mol·L^-1. Detection limit (3s/k) found by 10 parallel determinations of blank solutions and the standard deviation calculated was 2.0×10^-8mol·L^-1. Based on this phenomenon, a method of determination of peroxy acetic acid in disinfectant by fluorescence quenching of rhodamine B was proposed. The analyte solution was prepared by diluting 0.25 mL of the disinfectant with water to 500.0 mL after oxidation by adding 0.01 mol·L^-1 KMnO₄ solution dropwise until a stable and light pink color persisted in the solution. An aliquot of 1.0 mL of this solution was added to the reaction system (after the addition of KI solution), and kept the reaction solution standing for 15 min, then the analysis was carried on as described in the procedure. Three disinfectant samples from different factories was analyzed by the proposed method giving results in conformity with the labelled values given. Tests for recovery and precision were made by standard addition to the 3 samples as matrixes, giving results of recovery in the range of 96.5% to 97.5%, and RSDs (n=6) ranged from 1.3% to 2.6%. Somewhat lower recoveries were obtained due to instability of the peroxy acetic acid standard solution.