This study was aimed to establish ELISA for recombinant bovine IFN-γ (BovIFN-γ) detection and provide a new method for diagnosis of pathogenic infection. The total RNA was isolated from peripheral blood leucocytes cultured with PHA mitogen stimulation. Then bovine IFN-γ (BovIFN-γ) gene cDNA was amplified by RT-PCR and cloned into pET28a to obtain the expression plasmid designated as pETBovIFN-γ. The pETBovIFN-γ was further transformed into competent E. coli BL21 cells and a 18kD His-tagged protein as expected was expressed after IPTG induction. By using purified recombinant BovIFN-γ as antigen and lymphocyte-hybridoma technique, four hybridoma cell lines which stably secreted monoclonal antibodies against rBovIFN-γ were generated, designated as A7, A10, G6, and G10. The immunoglobin subset was identified as IgG₁. Western-blotting analysis and ELISA demonstrated that the monoclonal antibodies secreted by all the four hybridoma cell lines could react specifically to the recombinant BovIFN-γ, but not irrelative proteins such as Ag85B, ESAT-6-CFP-10 and GM-CSF, suggesting that the four hybridoma cell lines were rBovIFN-γ specific monoclonal antibodies. A sandwich ELISA was established by using A10 secreted monoclonal antibody and rabbit polyclonal antibodies against BovIFN-γ, HRP labeled goat anti-rabbit IgG. The results indicated that the sensitivity was 2ng/mL. This sandwich ELISA to detect BovIFN-γ paved the way to develop a sensitive method for specific infection detection such as bovine tuberculosis diagnosis.