Abstract: To study the extraction process of paeoniflorin in root bark of Paeonia delavayi var.lutea.The paeoniflorin was extracted by water, reflux extraction and ultrasonic extraction and the content was assayed by HPLC.Using orthogonal experiments analysis, the optimal extraction conditions of three extraction process were as follows:the water bath extraction method:50% ethanol, extraction time 2h, ratio of material to solvent 1∶450, extract 2 times.Content of the optimum conditions of extraction of paeoniflorin was 27.217mg/g.The reflux extraction method:50% ethanol, extraction time 2h, ratio of material to solvent 1∶450, extract 3 times.Content of the optimum conditions of extraction of paeoniflorin was 26.688mg/g.The ultrasonic cell crushing extraction method:90% ethanol, extraction time 30min, ratio of material to solvent 1∶450, extract 3 times.Content of the optimum conditions of extraction of paeoniflorin was 30.468mg/g.Methods established were simple, reliable and reproducible, which could provide data basis in a comprehensive study of root bark of P.delavayi var.lutea.