Two separate studies (study I and study II) were conducted to evaluate the efficacy of ELISA serological test for the detection of IgG antibodies against specific glycolipid antigen (PGLTb1) and ESAT 6 antigen of Mycobacterium tuberculosis, respectively. These results were compared with bacteriological tests [Ziehl Neelson (ZN) staining for acid-fast bacilli and culture on Lowenstein Jensen (LJ) medium] and polymerase chain reaction (PCR) targeting IS6110 sequence. Both studies were carried out on children with pulmonary, central nervous system, lymph node, and gastrointestinal tuberculosis along with matching controls (65 cases and 27 controls for study I and 83 cases and 22 controls for study II). Informed consents of their parents or guardians were taken. They were subjected to clinical examination, relevant laboratory investigations, tuberculin test and chest radiograph. Relevant body fluids were subjected to bacteriological tests and PCR. Sera samples were analyzed for antibodies against PGLTbl and ESAT 6 antigen in study I and study II, respectively. ELISA tests showed a significantly higher sensitivity (49% study I; 53%, study II) as compared with LJ medium culture method (15.4%, study I; 28.9% study II) and ZN staining (27.7%, study I; 20.5%, study II) in all patients (p < 0.05). The results were comparable with PCR (40%, study I; 42.2% study II). Specificity of ELISA test was 100% in all the patients except in those with pulmonary disease (92.8%, study I; 84.8%, study II). In view of the convenience, low cost and comparable sensitivity with PCR, these ELISA tests have a promising future in the diagnosis of childhood tuberculosis.