Objectives: Tuberculosis (TB) is curable if diagnosed correctly and promptly. However, the lack of effective and accessible point-of-care tests hindered the systematic screening of TB. The current TB diagnostic methods, including molecular tests, have failed to deliver the capacity needed in the endemic countries to restrict the ongoing pandemic. The detection of Mycobacterium tuberculosis by serology offers several advantages, including rapid and low-cost disease detection. Earlier, we had evaluated the diagnostic utility of five novel recombinant antigens, namely, SS-1, SS-2, SS-3, SS-4, and SS-5, with Indian patient sera. However, antibody detection has some limitations, and therefore, in the present study, we aimed to generate monoclonal antibodies and explore the utility of the most promising antibodies for the detection of TB. Materials and Methods: We used the three best recombinant antigens, that is, Rv2145c (SS-1), Rv1827 (SS-4), and Rv2970c (SS-5) for the generation of monoclonal antibodies. The monoclonal antibodies were developed using hybridoma technology. Further, the diagnostic utility of these monoclonal antibodies was evaluated in diagnosis of TB by sandwich enzyme-linked immunosorbent assay. Serum samples from bacteriologically confirmed TB cases and controls were used. Statistical Analysis: All statistical analysis was carried out using STATA-11.1 software (StataCorp LP, Texas, USA). The sensitivity and specificity were computed using an online tool (OpenEpi). Statistically significant differences between groups were defined as p<0.05. Results: A total of 384 serum samples were included in the study. This included 144 pulmonary TB cases, 68 extrapulmonary TB cases, 50 disease controls and 125 healthy controls. The sensitivity and specificity of our three monoclonal antibodies (mAb_SS-1, mAb_SS-4, and mAb_SS-5) for detecting all forms of TB ranged from 86.49% to 97.44% and 96.57% to 98.29%, respectively. The receiver operative characteristic curve showed a significant statistical difference between TB and healthy subjects (P < 0.001). Conclusions: Our data suggested that mAb_SS-1, mAb_SS-4, and mAb_SS-5 could be used as potential TB screening tests, especially in the resource-limiting setting.