Direct cloning and expression of large biosynthetic gene clusters (BGC, >30 kb) in  Bacillus subtilis typically involves a challenging process that requires assembly in  Saccharomyces cerevisiae, propagation in  Escherichia coli, and subsequent integration into  B. subtilis genome. To address these challenges, we developed an improved capture vector, pCAPB2S, by replacing the high-copy origin pUC ori with the low-copy oriV and oriS, and assembled with stability-relative elements sopA, sopB, and sopC, leading to an 80% positive rate for cloning the mycosubtilin BGC (~38 kb) from B. atrophaeus YK-3 in S. cerevisiae VL6-48, representing a 16-fold improvement over the original vector pCAPB2, and achieving high stability in E. coli. Furthermore, the intrinsic BsuM restriction-modification system in B. subtilis was knocked out, resulting in a transformation efficiency exceeding 60% for the vector carrying the mycosubtilin BGC (pCAPB2S- myc, >55 kb). HPLC-MS analysis confirmed that the result strain 1A751U- myc produced four homologs of mycosubtilin, which exhibited potent antimicrobial activity against a panel of pathogens and the phytopathogenic fungus Botrytis cinerea. The present study provides a powerful genetic tool for capturing large BGCs and demonstrates a strategy to improve transformation efficiency in B. subtilis.