Abstract: In order to obtain interferon-γ（IFN-γ） protein with antiviral activity, in this study, the coding region of buffalo IFN-γ gene was amplified by PCR, and the recombinant plasmid pET32 a-IFN-γ was constructed by linking it with pET-32 a vector. The recombinant plasmid pET32 a-IFN-γ was transformed into E.coli BL21 receptor cells, and the target protein was expressed by IPTG induction. SDS-PAGE analysis, purification, Western-blot identification, mass spectrometry analysis and antiviral activity detection were performed on the recombinant protein. The results showed that the PCR product size of buffalo IFN-γ coding region was about 520 bp. The recombinant plasmid pET32 a-IFN-γ was identified by double digestion and sequencing. The recombinant IFN-γ of the positive strain was about 39 ku in size, mainly expressed in soluble form, and could react specifically with His labeled murine monoclonal antibody. The amino acid sequence of the peptide was exactly matched with that of the target protein. The purified recombinant IFN-γ could effectively inhibit the cytopathic effect caused by bovine viral diarrhea virus on bovine kidney cells, and the antiviral activity was about 294.1 U/mg.These results indicated that buffalo IFN-γ protein was successfully expressed in E.coli and had certain antiviral activity.