Besides, WHO survey data from around the world shows that 0.6 million cases of multidrug-resistant tuberculosis (MDR-TB) were reported, of which about 0.24 million deaths occurred 2, 3, 4. About 10.0 million people with tuberculosis (TB) suffered in 2018, according to the World Health Organization (WHO) report. Mycobacterium tuberculosis is a global pathogen that infects about 1.5 billion individuals and kills 1.3 million individuals per year 1. Therefore, more experimental validations should be exposed to it. In conclusion, our findings show that the known multi-epitope vaccine may activate humoral and cellular immune responses and maybe a possible tuberculosis vaccine candidate. This research also sets out a good signal for the design of a peptide-based tuberculosis vaccine. coli, in-silico cloning and codon optimization were performed. For successful expression of the vaccine in E. The in silico immune simulation results showed significant response for immune cells. The constructed vaccine was used for further processing based on allergenicity, antigenicity, solubility, different physiochemical properties, and molecular docking scores. Bioinformatics tools were used to predict, refined, and validate the 3D structure and then docked with toll-like-receptor (TLR-3) using different servers. An adjuvant (Griselimycin) was also added to vaccine construct to improve its immunogenicity. By using the Immune Epitopes Database (IEDB), we predicted different B-cell and T-cell epitopes. In the current study, we select the very conserved, experimentally confirmed Mtb antigens, including Rv2608, Rv2684, Rv3804c (Ag85A), and Rv0125 (Mtb32A) to design a novel multi-epitope subunit vaccine. We applied advanced computational techniques to develop a universal TB vaccine. Bacillus Calmette–Guérin (BCG) is the single TB vaccine licensed for use in human beings and effectively protects infants and children against severe military and meningeal TB. Tuberculosis (TB) kills more individuals in the world than any other disease, and a threat made direr by the coverage of drug-resistant strains of Mycobacterium tuberculosis (Mtb).