A simple and sensitive fluorescent quenching method for the determination of trace hydrogen peroxide (H 2O 2) has been proposed to determine hydrogen peroxide in rain water sample. The method is based on the reaction of H 2O 2 with 3,3′-diethyloxadicarbocyanine iodide (DI) to form a compound which has no fluorescence in acetate buffer solution (pH 3.09). The maximum emission wavelength of the system is located at 604 nm with excitation at 570 nm. Under the optimal conditions, the calibration graph was obtained between the quenched fluorescence intensity and hydrogen peroxide concentration in the range of 5.0 × 10 −7 to 9.0 × 10 −4 mol L −1. The proposed method was applied to determine H 2O 2 in rain water samples, and the result was satisfactory. The mechanism involved in the reaction was also studied.