Brucellosis is a zoonotic caused by the which is a well-known infectious disease agent in domestic animals and if transmitted, it can cause infection in humans. Because brucellosis is contagious, its control depends on the eradication of the animal disease in farms. There are two vaccines based on the killed and/or weakened bacteria against and , but no recombinant vaccine is available for preventing the disease. The present study was designed to develop a multi-epitope vaccine against of and using virB10, Omp31 and Omp16 antigens by the prediction of T lymphocytes, T cell cytotoxicity and IFN-γ epitopes. 50S L7/L12 Ribosomal protein from Mycobacterium tuberculosis was used as a bovine TLR4 and TLR9 agonist. GPGPG, AAY and KK linkers were used as a linker. construct was well-integrated in the pET-32a Shuttle vector with I and III restriction enzymes. The final construct contained 769 amino acids, that it was soluble protein of about ∼82 kDa after expression in the SHuffle host. Modeled protein analysis based on the tertiary structure validation, molecular docking studies, molecular dynamics simulations results like RMSD, Gyration and RMSF as well as MM/PBSA analysis showed that this protein has a stable construct and is capable being in interaction with bovine TLR4 and TLR9. Analysis of the data obtained suggests that the proposed vaccine can induce the immune response by stimulating T- and B-cells, and may be used for prevention and remedial purposes, against and Communicated by Ramaswamy H. Sarma.