Bovine tuberculosis is an infectious and chronic disease affecting cattle, caused by Mycobacterium bovis (M. bovis). The zoonotic nature of the disease has a serious worldwide impact on human health. Also, the significant economic costs caused by such disease in addition to the deficiency of precise estimate of the actual disease prevalence necessitate more efficient detection and control measures, particularly in developing countries. The main target of the present work was to develop a local, less expensive bovine tuberculosis interferon-gamma ELISA (Bo-IFN-γ ELISA) kit for the rapid and accurate diagnosis of bovine tuberculosis. In the current study, three murine hybridomas producing monoclonal antibodies (mAbs) against bovine interferon-gamma (Bo-IFN-γ) were developed and their monoclonal antibodies were characterized. The produced mAbs were of IgM isotype and its specificity was proved using the western blot technique. The prepared mAbs were used for the development of the bovine IFN- γ ELISA test that was evaluated for laboratory diagnosis of bovine tuberculosis. The sensitivity and specificity of the developed ELISA kit as compared with the standard tuberculin skin test was determined. This method is based on the measurement of IFN-γ released from sensitized bovine lymphocytes upon exposure to the mycobacterial antigens. Using checkerboard titration, the optimal coating concentration of anti- Bo-IFN-γ was 20µg/well. Blood samples from apparently healthy cattle proved negative in the tuberculin test were examined with the developed kit and the cut-off value (COV) was equal to 0.30 optical density (OD). In a preliminary study for evaluation of the sensitivity and specificity of the developed ELISA, 23 cattle were examined with both the Bo-IFN-γ ELISA and the standard tuberculin skin test. The developed Bo-IFN-γ ELISA showed high sensitivity (98%) and specificity (71.4%) in the diagnosis of bovine tuberculosis as compared to the standard tuberculin skin test.