The invention relates to a method for extracting and separating bacitracin. The method is characterized by comprising the following steps of: (1) extracting and separating bacitracin concentrated liquor by ultrafiltration, namely acidizing bacitracin fermentation liquor, filtering or centrifuging by a vibrating screen to remove thalli, removing the thalli and macromolecular impurities by the ultrafiltration, removing an inorganic salt by resin adsorption and performing evaporation concentration under vacuum to obtain the bacitracin concentrated liquor; and (2) preparing a bacitracin crystalline solid by using the bacitracin concentrated liquor, namely performing production steps of extracting, decolorizing, nanofiltration, crystallizing and separating, drying, crushing, sieving, checking, the packaging of a finished product and the like on the bacitracin concentrated liquor to obtain the qualified bacitracin product. The method has the characteristics of high efficiency, simple equipment, high economy, large separation factor, high selectivity, low energy consumption, no secondary pollution, and the like, and is particularly suitable for the large-scale industrial production of the bacitracin; and operation can be performed continuously at the room temperature.