2-Hydroxy-1-naphthaldehyde thiosemicarbazon (HNT) had been synthesized and used as a new kind of substrate for horseradish peroxidase (HRP) in spectrofluorimetric determination of hydrogen peroxide (H(2)O(2)). The oxidation reaction of HNT with H(2)O(2) under the catalysis of HRP was studied in detail. The possible reaction mechanism was discussed. Under optimum experimental conditions, the oxidized product of HNT had excitation and emission maxima at 260 and 450 nm, respectively. The linear range of this method was 1.30 x 10(-9)-1.25 x 10(-5) mol l(-1) with a detection limit of 3.89 x 10(-10) mol l(-1). The effect of interferences, surfactants and organic solvents on the determination of H(2)O(2) had been investigated. A study to prove the existence of -O-O-H in PEGs was carried out. The proposed method was successfully applied to the determination of -O-O-H in polyethylene glycols.