Three phase partitioning was used to purify the polysaccharide from peony stamen and its physicochemical properties were analyzed. The optimal conditions of purification were obtained as follows: pH 7, ammonium sulfate concentration(w/v) 10% and ratio of t-butyl alcohol/crude extract(v/v).Under this condition, the polysaccharide recovery was 69% and the protein removal rate was 73%. The purity rate of the purified polysaccharide was 99.24%, and 95.36% pigment was removed by three phase partitioning. The Rresult of high performance liquid chromatography (HPLC) analysis showed that it was composed of rhamnose, galacturonic acid, glucose, galactose and arabinose, and the molar ratio was 1.24∶1.59∶2.00∶9.11∶1.68. The results of the Fourier transform infrared spectroscopy(FR-IR) and ¹H nuclear magnetic resonance (¹H NMR) showed that the polysaccharide from peony stamen was an acidic polysaccharide mainly composed of galacto-pyranose, and there are both α-and β-configuration, in which β-configuration accounted for a large proportion.