The invention discloses bacillus subtilis A16, which is preserved in China Center for Type Culture Collection (CCTCC) with CCTCC NO. 209105. The bacterial colony of the Bacillus subtilis A16 is oyster white on a beef extract peptone culture medium, has irregular shape and no luster, is not wet, has irregular edge, has diameter of 0.5-1cm, and has microfold surface, straight rod-shaped thallus and elliptic spore. The invention also discloses a preparation method and application of the bacillus subtilis A16. The bacillus subtilis A16 has broad bacteriostasis, can suppress exserohilum turcicum and helminthosporium maydis, has higher antagonism for exserohilum turcicum bacteria, helminthosporium maydis bacteria, maize curvularia leaf spot fungus, verticillium dahliae kleb, wheat scab pathogen, colletotrichum musae and banana vascular wilt bacteria, and has high prevention cure efficiency, and good safety and development application prospect.