Many diagnostic tools are essential for Mycobacterium bovis (M. bovis) eradication program. This study aimed to apply γ-IFN assay to detect bovine tuberculosis and multiplex PCR (m-PCR) for rapid identification of Mycobacterial isolates. A total no. of 150 cattle in 10 small farms at different Governorates in Egypt, were previously gave suspected results with comparative cervical tuberculin test (SICCT), they retested after 60 days later again with SICCT and bovine gamma-interferon (γ-IFN) immunoassay. Eighty-seven (58%) out of total 150 animals were +ve reactors by SICCT test while 80 (53.3%) animals gave +ve γ-IFN assay. The isolated M. bovis by conventional culturing and identification tests were +ve 55 (63.2%) out 87. The γ-IFN assay sensitivity and specificity gave 82.9% and 93.8% respectively. For rapid identification of different mycobacterial isolates using m-PCR two set of primers were used. The first set gave 123bp DNA PCR product expressing IS6110 insertion element for Mycobacterium tuberculosis (MTBC). The other one gave 500bp from RvD1Rv2031c genomic sequence definite to M. bovis. M-PCR findings were in a concordance with results of conventional culturing and identification tests with high sensitivity and specificity (100%). From this study, it is concluded that diagnosis of bovine tuberculosis (BTB) used tuberculin test and γ-IFN assay with m-PCR for rapid identification M. bovis isolates in living herds.