Mycobacterium tuberculosis (Mtb) is the pathogen that causes tuberculosis and develops resistance to many of the existing drugs. The sole licensed TB vaccine, BCG, is unable to provide a comprehensive defense. So, it is crucial to maintain the immunological response to eliminate tuberculosis. Our previous in silico study reported five uncharacterized proteins as potential vaccine antigens. In this article, we considered the uncharacterized Mtb H37Rv regions of difference (RD-2) Rv1987 protein as a promising vaccine candidate. The vaccine quality of the protein was analyzed using reverse vaccinology and immunoinformatics-based quality-checking parameters followed by an ex vivo preliminary investigation. In silico analysis of Rv1987 protein predicted it as surface localized, secretory, single helix, antigenic, non-allergenic, and non-homologous to the host protein. Immunoinformatics analysis of Rv1987 by CD4 + and CD8 + T-cells via MHC-I and MHC-II binding affinity and presence of B-cell epitope predicted its immunogenicity. The docked complex analysis of the 3D model structure of the protein with immune cell receptor TLR-4 revealed the protein's capability for potential interaction. Furthermore, the target protein-encoded gene Rv1987 was cloned, over-expressed, purified, and analyzed by mass spectrometry (MS) to report the target peptides. The qRT-PCR gene expression analysis shows that it is capable of activating macrophages and significantly increasing the production of a number of key cytokines (TNF-α, IL-1β, and IL-10). Our in-silico analysis and ex vivo preliminary investigations revealed the immunogenic potential of the target protein. These findings suggest that the Rv1987 be undertaken as a potent subunit vaccine antigen and that further animal model immuno-modulation studies would boost the novel TB vaccine discovery and/or BCG vaccine supplement pipeline.