The invention discloses bacillus subtilis NB12, as well as a culture method and an application thereof. The strain with a preservation number of CGMCC No.5383 is preserved in China General Microbiological Culture Collection Center on October 24, 2011. Researches prove that: the strain has strong inhibition effects to rhizoctonia solani, colletotrichum musae, pyricularia grisea, fusarium graminearum, fusarium oxysporum f.sp.cubense, peronophythora litchi, ustilago scitaminea, fulvia fulva, xanthomonas oryzae and xanthomonas oryzae pv.oryzicola, is a broad-spectrum antagonistic strain, can be used for prevention and control of various plant diseases, has a particular good prevention and control effect to the rice sheath blight and damping off in seedling phase of cowpea, has the advantages of harmlessness to human and livestock, greenness and safety, good environment compatibility, difficult resistance generation and the like, can be used for commercial development, and has a wide application prospect.