Abstract: To establish a method for preparation of high purity β-asarone and α-asarone from Acorus tatarinowii Schott by high pressure semi-preparative liquid chromatography, its volatile oil was prepared from Acorus tatarinowii Schott, and a semi-preparative high performance liquid chromatography was used to refine and purify β-asarone and α-asarone with a solvent composed of methanol and water ( 65∶ 35, v/v) .The structures of product were identified by EI-MS, 1H-NMR and13C-NMR, and their purities reached 99.43% and 99.08%, respectively. The method is easily operated, with high purity and preparation amount.It is suitable for high purity β-asarone and α-asarone reference material preparation from Acorus tatarinowii Schott.