Aqueous polymethylmethacrylate (PMMA)-capped CdSe/ZnS quantum dots were used as fluorescence probes for paeonol determination. Based on the fluorescence quenching of aqueous CdSe/ZnS quantum dots caused by paeonol, a simple, sensitive and rapid method was developed. Under the optimal conditions, with excitation and emission wavelengths at 350nm and 620nm, respectively, the calibration plot of F 0 −F with concentration of paeonol was linear in the range of 25.04–175.2mgL −1 with correlation coefficient of 0.9986. The limit of detection was 0.017mgL −1 . The concentration of paeonol in paeonol ointment was determined by the proposed method and the result agreed with the claimed value. Furthermore, the possible fluorescence quenching mechanism was discussed.