[Objective] To extract β-elemene from worst weed Eupatorium adenophorum Spreng, identify its structure and determine its content. [Method] Having obtained volatile oil from E. adenophorum by “water+glycol” distillation, β-elemene was separated by column chromatography with slica, silver nitrate modified slica, D101 and HP20 macroporous resin respectively. The pure product of β-elemene was prepared by HPLC, and its molecular structure was validated by UV, IR, 1H NMR, 13C NMR and HRMS. [Result] The purity and yield of product was up to 63.1% and 75.6% respectively if using HP20 column chromatography, the optimal one for separation of β-elemene from E. adenophorum. Through three steps of “water+glycol” distillation, HP20 column chromatography and HPLC preparation, the β-elemene product with 98.6% purity and 36.5% yield could be obtained from E. adenophorum. [Conclusion] HP20 column chromatography is the most efficient way to separate β-elemene from E. adenophorum, which has the advantages of low cost and easy operation. [Objective] To extract β-elemene from worst weed Eupatorium adenophorum Spreng, identify its structure and determine its content. [Method] Having obtained volatile oil from E. adenophorum by “water + glycol” column chromatography with slica, silver nitrate modified slica, D101 and HP20 macroporous resin respectively. The pure product of β-elemene was prepared by HPLC, and its molecular structure was validated by UV, IR, 1H NMR, 13C NMR and HRMS. ] The purity and yield of product was up to 63.1% and 75.6% respectively if using HP20 column chromatography, the optimal one for separation of β-elemene from E. adenophorum. Through three steps of “water + glycol ” distillation, HP20 column chromatography and HPLC preparation, the β-elemene product with 98.6% purity and 36.5% yield could be obtained from E. adenophorum. [Conclusion] HP20 column chromatography is the most efficient way to separate β-elemene from E. adenophorum, which has the advantages of low cost and easy operation.