Increased incidence of extrapulmonary tuberculosis (EPTB) as co-infection is observed due to rise in human immunodeficiency virus (HIV) infection. However, the precise diagnosis of EPTB is faced with difficulty due to the lack of tissue biopsy or fine-needle aspiration cytology (FNAC) facilities in rural hospitals. Enzyme linked immunosorbent assay (ELISA) will be simple, convenient and doesn’t require sophisticated laboratory. Mycobacterial antigens ES-31, ES-43 and EST-6 antigens were isolated from Mycobacterium tuberculosis (M. tuberculosis) H37Ra bacilli by affinity chromatography. Cocktail of these antigens and their affinity purified antibodies were explored for detection of antibody and antigen by Indirect and Sandwich ELISA respectively. In a preliminary study with bacteriologically confirmed EPTB cases (n=32), assay of antibody/free antigen/immunecomplexed (IC) antigen showed a sensitivity of 100% and specificity of 90%. Based on this study, sera from suspected EPTB patients (n=164) diagnosed by clinical and other laboratory investigations, non-tubercular disease patients (n=75) and healthy controls (n=75) were screened. A sensitivity and specificity of 72% and 91% for antibody detection, 70% and 94% for circulating free antigen and 63% and 98% for circulating IC antigen detection were observed. On combining the positivity of antibody, circulating free and IC-antigen, overall sensitivity of 96% and specificity of 91% were observed in EPTB. Tuberculous antibody detection to cocktail antigen was found to be useful in detection of EPTB. However, circulating free and IC-antigen detection may be a better marker for detection of different groups of EPTB.