The invention discloses a method for preparing pneumocandin B0 by using a fermentation culture product of filamentous fungi glarea lozoyensis. According to the method, a filter aid is adopted; with steps such as leaching, discoloring, adsorption, crystallization, and the like, a pneumocandin B0 crude extract is obtained; and the crude extract is subjected to chromatographic separation by using polymer microspheres, such that the high-purity pneumocandin B0 product is obtained. The method provided by the invention has the advantages that: pneumocandin B0 is extracted and separated by using macroporous resin; the process is simple and feasible, and is suitable for industrialized productions; the polymer microspheres are used in chromatographic separation of the product for a first time, and high-purity pneumocandin B0 product can be prepared.