Objective To establish a high performance liquid chromatography (HPLC) method for the determination of paeoniflorin in a natural Chinese herbal medicine. Methods The chromatographic column was${\mathrm{C}}_{18}$column (${250}\mathrm{\;{mm}}\times$ ${4.6}\mathrm{\;{mm}},{5\mu }\mathrm{m}$), the mobile phase was acetonitrile -0.05% phosphoric acid aqueous solution $\left({{20}: {80}, V/V}\right)$, the flow rate was ${0.8}\mathrm{\;{mL}}/\mathrm{{min}}$, the column temperature was ${30}^{\circ }\mathrm{C}$, the detection wavelength is ${237}\mathrm{\;{nm}}$, and the injection volume was${10\mu }\mathrm{L}$. Results The linear range of paeoniflorin was ${24.68}\sim {246.8\mu }\mathrm{g}/\mathrm{{mL}}$, the linearity of paeoniflorin was good $\left({{r}^{2}= {0.9999}}\right.$, $n = 5$), the results of precision, stability and repeatability experiment were good, and the RSD values were less than $2\%$, the average recovery was ${102.0}\%$, RSD was ${1.49}\%\left({n = 9}\right)$. Conclusion The method is simple, convenient, easy to operate, and with strong specific, good separationand, good repeatability. It is suitable for the determination of paeoniflorin in natural Chinese herbal medicine, and provides a reference for the determination of paeoniflorin in compound preparations with Radix Paeoniae rubra as raw material.