The invention relates to a preparation method of total glucosides from Paeonia lactiflora Pall., and a Paeonia lactiflora Pall. total glucoside preparation and use thereof. The method comprises: grinding, extracting, concentrating under reduced pressure, extracting, decolorizing, filtering, re-concentrating and drying. Column chromatography also can be used. The preparation method provided by theinvention greatly improves the content of total glucosides from Paeonia lactiflora Pall. to over 80 percent. The total glucosides from Paeonia lactiflora Pall., which is provided by the invention, can be used in the preparation of medicines for treating gynecological diseases, in particular the use in the preparation of medicines for treating dysmenorrheal, abnormal menstruation or endometriosis.The preparation method simplifies operation, adopts extraction solvent with low toxicity, allows for easy solvent recovery, is friendly to human beings and the environment and in accordance with environment protection requirements.