Bovine interferon-gamma (b-IFN-γ) is a key cytokine biomarker, the activity of which increases during chronic animal infection with intracellular pathogens. Disease diagnosis can be performed based on detection of the b-IFN-γ blood levels in infected animals via specific antibodies. The production of specific reagents for b-IFN-γ identification involves the gene cloning of native templates followed by protein expression, and often produces misfolded proteins due to improper translation by heterologous cell platforms. In this study, a computational approach combined with gene engineering expression technology was used for the bacterially production of a recombinant b-IFN-γ yielding 2 µg/ml soluble protein. The rb-IFN-γ was recognized by Western blot with specific monoclonal antibodies and bovine IFN-γ-capture ELISA. The recombinant cytokine demonstrated good in vivo immunogenic properties, raising mouse polyclonal antibodies titers of above 51.2K, and allowing recognition of blood plasma b-IFN-γ. The synthesized bovine cytokine is expected to aid in developing b-IFN-γ specific immunoassays for diagnosing bovine infectious diseases.