The invention discloses a separation and purification method of antifungal active substances in S.lydicus fermentation liquor. The method comprises the steps of (1) centrifuging the fermentation liquor of S.lydicus which has the collection number of CGMCC No.3075, collecting supernatant, adding a flocculant, uniformly mixing, standing, and filtering in vacuum to obtain filter liquor; (2) washing the filter liquor on a macroporous resin chromatographic column with methyl alcohol to remove pigment and impurities which are not adsorbed, and then eluting active components with an ethyl alcohol aqueous solution; (3) mixing active eluants and concentrating to obtain concentrated liquor; and (4) carrying out molecular sieve chromatography on the concentrated liquor by virtue of a glucan gel chromatographic column, eluting with the ethyl alcohol aqueous solution, collecting eluants in different tubes, numbering the elutants in sequence, respectively detecting the antifungal activities of the elutants, and collecting antifungal active components. According to the invention, the separation and purification processes are simple and rapid, convenient to operate and low in cost; three-waste emission is little; the purification process is mild, and the purities of the active substances are relatively high; the eluants are low in toxicity and simple to recycle.