Abstract: To investigate the antibacterial substances from Bacillus amyloliquefaciens HE and antibacterial characteristics, we synthetized the genes related to lipopeptides synthesis by PCR detection according to the stability of B. amyloliquefaciens fermentation broth, and further analyzed the active substances by UPLC-Q-TOF-MS. Then we applied the inhibition zone method to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of lipopeptide against Aeromonas hydrophila, whose antimicrobial activity was discussed from the microscopic structure. The antimicrobial active substances were stable against high temperature, acid, acid-base and protease. The PCR with four primer pairs were cloned and sequenced. The BLAST analysis shows that the ituA, fenB, sfP and mycB genes existed in the genome of B. amyloliquefaciens HE. Three kinds of lipopetides (Surfactin, Iturin and Fengycin) were identified in the antimicrobial extract by mass spectrum analysis. The MIC and MBC of antimicrobial lipopetides against A. hydrophila were both 137.97 μg·mL⁻¹ by inhibition zone method. The microscopic characteristics indicate that lipopeptides can cause membrane collapse and holes in A. hydrophila cells. The results provide references for the structural identification and safety evaluation of active metabolites of B. amyloliquefaciens.