Early detection of bovine tuberculosis in infected cattle is important to eliminate the infected animals from a herd, and to reduce the risk post by animals and animal products to human.  Methods for the detection of mycobacteria are continuously being developed. A total of 1050 cows were tested by tuberculin test and the lymph nodes and internal organs of tuberculin positive cows were examined by culture method and confirmed by RT- PCR. The whole blood samples that collected as soon as the reading of the tuberculin test from both the tuberculin positive and tuberculin negative cows were used for detection of gamma interferon release (bovigam, prionics). The obtained results revealed that 57 cows were positive tuberculin test (5.4%), 37 out of them were harbored the acid-fast bacilli of mycobacteria (65 %), which confirmed using RT -PCR, while there are 49 out of 57 tuberculin positive cows were tuberculous by gamma interferon assay (86 %).  There are 3 cases out of 57 tuberculin negative cows were tuberculous by gamma interferon assay. The use of gamma interferon assay has the superiority for diagnosis of bovine tuberculosis over the conventional culture method and tuberculin test due to the obstacles of the decontamination process as well as the field problems of tuberculin test.