The invention provides a method for separation and extraction of epothilone B from a sorangium cellulosum fermentation liquid, and belongs to the microbial fermentation pharmacy field. The method comprises the steps: after shake-flask cultivation of sorangium cellulosum strains, amplifying step by step, fermenting sorangium cellulosum, followed by carrying out macroporous resin adsorption, ethyl acetate desorption and methanol redissolution, and thus obtaining a crude product; carrying out column chromatography and HPLC analysis of the crude product to obtain a preliminary component of epothilone B, then adding a molecularly imprinted polymer for specific adsorption of epothilone B, and finally carrying out methanol elution and crystallization to obtain epothilone B. Sorangium cellulosum is produced by step-by-step fermentation, and at the same time, the molecularly imprinted polymer with the specific targeted adsorption capacity is adopted in the separation and extraction process, not only has a strong adsorption capacity on epothilone B, but also can easily extract epothilone B, so as to reduce the extraction cost of separation and extraction of epothilone drugs, and have more wide industrialized application prospects and economic benefits.