The fluorescence quenching effect of paeonol on the cerium(III) and sodium dodecyl sulfate (SDS) sensitization effect of system were investigated. A new method for paeonol analysis has been established based on the fluorescence intensity quenching of cerium(III) in SDS medium. With excitation and emission wavelengths at 258 nm and 361 nm, respectively, at pH 3.0, the linear range of calibration curve for the determination of paeonol was 0.50 μg/mL-10.00 μg/mL with correlation coefficient of 0.9987. The detection limit was 0.09 g/mL and RSD was 1.69% (n = 3, c = 2.50 μg/mL). The method has been applied for the determination of paeonol in real samples with satisfactory results. The recoveries of the samples ranged from 96.5% to 99.8%. Furthermore, the cerium(III) fluorescence quenching mechanism was discussed by Stern-Volmer equation and fluorescence quantum yield.