Abstract: Screening highly efficient antagonistic bacteria and exploring their natural antibacterial substances are one of the important ways to develop precise biological control drugs against the pathogen Vibrio harveyi and achieve green mariculture. In this experiment, the plate confrontation method was used to screen antagonistic bacteria with significant antagonistic effects against V. harveyi; the optimal fermentation conditions and stability of the antibacterial substances in the supernatant were explored; and the crude antibacterial proteins were precipitated by ammonium sulfate with a saturation. The results showed that Bacillus velezensis BS2 - 7 had the most significant antagonistic effect against the aquaculture pathogen V. harveyi (GDH11385); the fermentation medium and fermentation temperature had no significant effect on the antibacterial activity, while the fermentation duration had a significant effect (P < 0.05). The supernatant had a wide antibacterial spectrum and showed antagonistic activity against V. harveyi, V. owensii, Vibrio parahaemolyticus, and Bacillus altitudinis; temperature, pH, and ultraviolet irradiation had significant effects on the antibacterial activity of the antibacterial substances in the supernatant (P < 0.05). The crude proteins separated from the fractions with ammonium sulfate saturation of 10% and 20% had antibacterial activity against V. harveyi and V. owensii. This study can provide sample support and theoretical basis for the development of antibacterial protein drugs for the biological control of Vibrio pathogens.