Antimicrobial agents play an important role in the prevention and treatment of bacterial infections. However, the extensive use of antimicrobial agents, particularly the misuse of antibiotics in the aquatic animal breeding, has led to various issues, such as antibiotic resistance, pollution of breeding environments, and presence of drug residues in livestock, which need to be addressed urgently. In this paper, the effects of exogenous L-cysteine on the susceptibility of Aeromonas hydrophila to florfenicol were studied. The in vitro bactericidal assay found that 4.00 mmol/L L-cysteine could significantly improve the bactericidal effect of florfenicol by 1.9-11.1 times and that the bactericidal effect increased with increasing L-cysteine concentration and exposure time. Tissue bacterial eradication experiments showed that the combination of L-cysteine and florfenicol could significantly eliminate A. hydrophila in liver, spleen and kidney tissue by 4-16 times compared to florfenicol alone. Oral administration or injection with L-cysteine or/and florfenicol showed that the combination of L-cysteine and florfenicol greatly increased the survival rate of infected fish. Moreover, we also found L-cysteine played a certain role in tissue bacteria eradication and fish immune protection. At last, we found that 1.00-4.00 mmol/L L-cysteine increased the susceptibility of A. hydrophila to florfenicol by 2-4 times. We speculate that L-cysteine may enhance the susceptibility of A. hydrophila to florfenicol and increase the bactericidal effect of florfenicol. In summary, L-cysteine as a synergistic agent of florfenicol, can not only improve the bactericidal effect, but also significantly reduce the use of antimicrobial agents. Therefore, we present a novel approach in fighting against A. hydrophila with practical application value.