To extract and isolate β-elemene from worst weed Eupatorium adenophorum Spreng, identify its structure and determine its content. Having obtained volatile oil from E. adenophorum by “water+ glycol” distillation, β-elemene was isolated by column chromatography with HP20 macroporous resin, then 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. 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, and it’s molecular structure can be validated. “Water + glycol distillation” is the better way to extraction volatile oil from E. adenophorum than traditional water distillation. HP20 column chromatography is the more efficient way to isolate β-elemene from volatile oil than previous silver ion coordination chromatography.