Tuberculosis is an airborne transmissible disease caused by Mycobacterium tuberculosis that infects millions of lives worldwide. There is still no single comprehensive therapy or preventative available for the lethal illness. Currently, the available vaccine, BCG is ineffectual in preventing the prophylactic adult pulmonary TB and reactivation of latent tuberculosis. Therefore, this investigation was intended to design a new multi-epitope vaccine that can address the existing problems. The subtractive proteomics approach was implemented to prioritize essential, virulence, druggable, and antigenic proteins as suitable vaccine candidates. Furthermore, a reverse vaccinology-based immunoinformatics technique was employed to identify potential B-cell, helper T lymphocytes (HTL), and cytotoxic T lymphocytes (CTL) epitopes from the target proteins. Immune-stimulating adjuvant, linkers, and PADRE (Pan HLA-DR epitopes) amino acid sequences along with the selected epitopes were used to construct a chimeric multi-epitope vaccine. The molecular docking and normal mode analysis (NMA) were carried out to evaluate the binding mode of the designed vaccine with different immunogenic receptors (MHC-I, MHC-II, and Tlr4). In addition, the MD simulation, followed by essential dynamics study and MMPBSA analysis, was carried out to understand the dynamics and stability of the complexes. In-silico cloning was accomplished using E.coli as an expression system to express the designed vaccine successfully. Finally, the immune simulation study has foreseen that our designed vaccine could induce a significant immune response by elevation of different immunoglobulins in the host. However, there is an imperative need for the experimental validation of the designed vaccine in animal models to confer effectiveness and safety.HIGHLIGHTSMulti-epitope based vaccine was designed against Mycobacterium tuberculosis using subtractive proteomics and Immunoinformatics approach.The vaccine was found to be antigenic, non-allergenic, immunogenic, and stable based on in-silico prediction.Population coverage analysis of the proposed vaccine predicts an effective response in the world population.The molecular docking, MD simulation, and MM-PBSA study confirm the stable interaction of the vaccine with immunogenic receptors.In silico cloning and immune simulation of the vaccine demonstrated its successful expression in E.coli and induction of immune response in the host. Communicated by Ramaswamy H. Sarma.