To study the bacteriostatic effect of triple yolk antibody (IgY) against Vibrio harveyi, V. alginolyticus and V. parahaemolyticus in vitro, the triple inactivated vaccine of V. harveyi, V. alginolyticus and V. parahaemolyticus was prepared and the laying hens were immunized. The eggs were collected for the preparation of IgY and their titer, purity and specificity were determined. By immunofluorescence, scanning electron microscopy and in vitro antibacterial experiments, the in vitro antibacterial effect of the prepared triple IgY on three species of Vibrio was preliminarily investigated. The results showed that the ELISA detected a titer of up to 1∶51 200 for the IgY, which it maintained for 5 weeks; SDS-PAGE analysis of the isolated and purified IgY showed that the protein impurity bands in the IgY were gradually reduced, and the purity increased with purification. Immunofluorescence and immunoblotting experiments demonstrated high specificity of binding of the specific IgY to the antigen. Further observation by scanning electron microscopy showed that, compared to Vibrio treated with non-specific IgY, those treated with specific IgY adhered to each other, the surface of the bacterial cells appeared rough, and the cell wall was damaged. In vitro bacterial inhibition assays showed that the specific IgY significantly inhibited Vibrio in a dose-dependent manner. In summary, the triple IgY prepared in this study was able to specifically bind to V. harveyi, V. alginolyticus and V. parahaemolyticus, and it was found through in vitro experiments that the IgY could play an inhibitory role by destroying the integrity of the bacterial cells through the agglutination and adhesion.