In this paper, a sensitive and simple detecting system was developed for quantitative analysis of both hydroquinone (H 2 Q) and hydrogen peroxide (H 2 O 2 ), based on the successful combination of horse radish peroxidase (HRP) and water-soluble conjugate fluorescence polymers PPESO 3 . In the presence of HRP and H 2 O 2 , H 2 Q could be oxidized to 1,4-benzoquinone (BQ), an intermediate, which plays the main role in the enhanced quenching of the photoluminescence (PL) intensity of PPESO 3 . The quenching PL intensity of PPESO 3 (I 0 /I) was proportional to the concentration of H 2 Q and H 2 O 2 in the range of 1.0×10 −6 to 2.0×10 −3 mol/L (R 2 =0.996) and 6.0×10 −6 to 2.0×10 −3 mol/L (R 2 =0.999), respectively. The detection limit for H 2 Q and H 2 O 2 was 5.0×10 −7 mol/L and 1.0×10 −6 mol/L, respectively. The present fluorescence quenching method was successfully applied for the determination of H 2 Q in the lake water, rainwater, tap-water and chemical plant wastewater samples. Compared with previous reports, the fluorescence quenching approach described in this work is simple and rapid with high sensitivity, which has a potential application for detecting various analytes which can be translated into quinone.