A method for preparation of crenulatin reference substance was developed in this paper. Crenulatin was extracted from the roots of Rhodiola Crenulata. H by alcohol, isolated by macroporousresin, purified by column chromatography, and structurally analyzed by spectroscopy. GC-MS analysis showed that the contents of crenulatin were higher than 99%. The achieved crenulatin could be used as reference substances for the qualitative and quantitative analysis of the raw materials and products of Rhodiola Crenulata. H.