Vibrio scophthalmi and Aeromonas salmonicida can cause high turbot mortality and huge economic losses. Presently, vaccination is the most promising method for preventing communicable diseases. In this study, we used formalin to kill V. scophthalmi and A. salmonicida cells, and mixed with the mineralized oil adjuvant (Montanide™ ISA 763 AVG) to prepare the bivalent inactivated vaccine. The results showed that turbot inoculated with the bivalent inactivated vaccine exhibited strong tolerance to the infection of V. scophthalmi and A. salmonicida, and no obvious clinical symptoms and pathological changes were observed. The activities of enzymes lysozyme, acid phosphatase and complement C3 had significantly increased after the vaccination. The antibody titer response of vaccinated turbot was greatly boosted, which was positively connected with the immunological impact according to ELISA results. Simultaneously, the expression levels of immune-related genes such as MHC-IIα, MHC-IIβ, CD4, CD8, TNF-α and IL-1β were up-regulated, demonstrating that it might stimulate humoral and cellular immunological response in turbot. These findings highlight the potential of the bivalent inactivated vaccine for controlling V. scophthalmi and A. salmonicida infections in turbot.