Abstract: OBJECTIVE To establish the method of separation, purifying and idenfication method of paeoniflorin, and to elevate effectually paeoniflorin content. METHODS Paeonia veitchii Lynch was extracted with ethanol-water solution, separated and purified by AB-8 macroporous resin and preparative HPLC. The structure was identified on the basis of physical and chemical characters and spectral analysis and quantitatively determined by HPLC. RESULTS Paeoniflorin with purity over 95.4%, can be prepared with the total recovery more than 81.03%. CONCLUSION High purity products can be obtained with this process which is high-performance, convenient, low cost and high recovery and is a good method of separation and purification of paeoniflorin.