Abstract: In order to explore the optimal fermentation conditions and stability of the antibacterial substances in the supernatant, the plate confrontation method was used to screen antagonistic bacteria with the significant antagonistic effects against V. harveyi , and the salting method with the different ammonium sulfate saturation was performed to extract the crude antibacterial proteins. The results showed that Bacillus velezensis BS2-7 had the most significant antagonistic effects against the aquaculture pathogen V. harveyi (GDH11385), the fermentation medium and fermentation temperature had no significant effects on the antibacterial activity, while the fermentation duration had a significant effect (P<0.05); the supernatant had a wide antibacterial spectrum and showed the antagonistic activity against V.harveyi, V.owensii, Vibrio parahaemolyticus, and Bacillus altitudinis; the temperature, pH, and ultraviolet irradiation had significant effects (P<0.05); when the ammonium sulfate with 10% saturation and 20% saturation were added into the supernatant, the crude proteins separated from the fractions had the antibacterial activity against V. harveyi and V.owensii. Our findings provide the sample support and the theoretical basis for the development of antibacterial protein drugs against Vibrio pathogens.