Abstract: Scale-up production technology of highly purified bacillomycin D was established to support the exploration for its agriculturally antibiotic activity. After isolation, purification and molecular identification, the bioactive compounds extracted from Bacillus amyloliquefaciens TYg 3-2 were identified as antibiotic lipopeptides bacillomycin D. The refined nature media and chemically defined available carbon and nitrogen sources, including yeast extract, peptone, glucose, and (NH4)2SO4, were combined and optimized using response surface method for high-yield complex medium of bacillomycin D. Using the optimized medium, the yield of bacillomycin D in 5 L bioreactor reached 990 mg/L. The productivity reached 20.62 mg/(L·h), which was much higher than the previously reported productivity of 10.23 mg/(L·h). Biomass-supernatant separation, acid precipitation, methanol extraction, Et2O precipitation, macroporous absorption resin column chromatography and silica gel column chromatography were conducted for efficient purification of bacillomycin D. And 94.72% purity was obtained when high-quality raw material was used. Preliminary exploration for antibiotic activity was conducted using the highly purified bacillomycin D sample. Inhibition tests in vitro demonstrated that bacillomycin D was the main effective substance against Drechslera turcica, Curvularia lunata, Ustilaginoidea virens and Alternaria longipes in vitro. And the result of pot test in vivo revealed that bacillomycin D was the promotor on biocontrol efficacy of homologous B. amyloliquefaciens TYg 3-2 biomass. Because the mixture of 500 mg/L bacillomycin D and 4×106 CFU/mL B. amyloliquefaciens TYg 3-2 biomass exhibited 95.3% biocontrol efficacy against Colletotrichum orbiculare in vivo, which was higher than those of bacillomycin D alone (78.4%) and Bacillus biomass alone (0). The potential value of bacillomycin D in the field of agricultural antibiotic was preliminarily explored. And scale-up production technology of highly purified lipopeptides played an important role in this exploration. Not only could it isolate and purify target lipopeptide from fermentation broth for this agricultural antibiotic research without the loss in producing bacteria (the spore of which was the feedstock of biocontrol agent), but also can it satisfy the requirement of toxicological tests of pesticide registration in China (sample with 85% purity in grams). As a result, this research was able to support the research and development of agriculturally antibiotic value in Bacillus lipopeptides.