Tuberculosis (TB) causes considerable morbidity among millions of people each year worldwide and ranks as the second leading cause of death from an infectious disease. The present anti-TB drugs produce side-effects primarily liver damage and other adverse reactions like nausea, vomiting, and anorexia. The increase in multi-drug resistant strains of M. tuberculosis has decreased the effectiveness of current standard Tuberculosis treatment options. Thus, the discovery of anti-tuberculosis agents that target new pathways with novel mechanisms of action is crucial for effective short-term tuberculosis therapy that will limit the development of resistance. Tubulosine, an isoquinoline alkaloid from the stem bark of Alangium lamarckii reported to exhibit antiplasmodic activity. The present study was designed to perform molecular docking analysis of Tubulosine for find the binding conformation, affinity and orientation of Tubulosine in the active sites of Mycobacterium tuberculosis DNA gyrase, Isocitrate lyase, Thymidine monophosphate kinase which can inhibit the Tuberculosis. The crystal structure of M. tuberculosis DNA gyrase (4G3N), Isocitratelyase (1F61), Thymidine monophosphate kinase (1W2G) were obtained from the Protein Data Bank (RCSB PDB). The PDB file was prepared using the software UCSF Chimera. The Molecular docking analysis has shown Tubulosine to potentially inhibit thymidine monophosphate kinase (-9.36kcal/mol) than DNA gyrase and Isocitratelyase at a very minimal concentration of 138.37nM. Thus this study offers a route for the development of Tubulosine against multidrug resistant tuberculosis and need further in-vitro investigations to confirm their efficacy and drug ability.