Bacillus amyloliquefaciensis an interesting microbe in the food processing and manufacturing industries. Non-coding small RNAs (sRNAs) have been shown to play a crucial role in the physiology and metabolism of bacteria by post-transcriptionally regulating gene expression. This study investigated the function of novel sRNA FenSr3 by constructingfenSr3deficient strain and complementary strains inB. amyloliquefaciensLPB-18 , which were named LPN-18N and LPB-18P, respectively. The result showed significant differences in fengycin yield between strain LPB -18N and LPB-18P. The production of fengycin was significantly enhanced inB. amyloliquefaciensLPB-18N, compared with that of the strain LPB-18 from 190.908 mg/L to 327.598 mg/L. Moreover, the production of fengycin decreased from 190.464 mg/L to 38.6 mg/L inB . amyloliquefaciensLPB-18P. A comparative transcriptome sequencing was carried out to better understand the complex regulatory mechanism. Transcription analysis revealed that 1037 genes were differentially expressed betweenB. amyloliquefaciensLPB-18 andB. amyloliquefaciensLPB-18N, including the key regulatory genes in fatty acid, amino acid biosynthesis, and central carbon metabolism, which could provide sufficient quantities of building precursors for fengycin biosynthesis. The biofilm formation and sporulation was also enhanced in the strain LPB-18N, which indicates that FenSr3 could play a vital role in stress resistance and promotes survival inB. amyloliquefaciens. Some sRNAs involved in stress response have been identified in the literature, but their regulatory roles in fengycin production remain unclear. The study will contribute a novel perspective to the regulation mechanism of biosynthesis and the optimization of key metabolites ofB. amyloliquefaciens.