Abstract: In order to extract and isolate an agro-antibiotic from the fermentation products from a soil rare actinomycetes strain, acetone was chosen as extracting agent instead of more commonly used methanol as extracting agent and ethyl acetate as column chromatography eluent. A 3 levels and 3 factors orthogonal test design method was used to optimize extraction technology. From the data processing results by extremum difference analysis and variance analysis method, the optimal extraction condition was to extract with 3 fold acetone with 2 hours. Silica gel column chromatography was used to separate the active ingredients and ethyl acetate as eluent. A large amount of impurities was removed by once column chromatography and the recovery was more than 83%, which built foundation for further separation and purification of effective components.