In order to obtain the monoclonal antibody of gI protein of infectious bovine rhinotracheitis virus（IBRV），a recombinant prokaryotic expression plasmid pET32a-gI with truncated gI gene was constructed，induced，expressed and purified，the expression of gI protein in E.coli. was verified by SDS-PAGE and Western blot. BALB/C mice were immunized with purified gI protein，and then the monoclonal cell line gI14 targeting IBRV gI was obtained by cell fusion，screening and sub-cloning. Monoclonal antibody ascites against IBRV gI protein was prepared by the in vivo induction method to test and verify the specificity of IBRV gI monoclonal antibody fluid by Western blot and indirect immunofluorescence assay（IFA）. The results showed that，by Western blot，the relative molecular weight of the prokaryotic expressed gI fusion protein was 34 ku，the monoclonal antibody against gI protein reacted well with the gI expression protein，and failed to specifically bind to pET-32a（+）empty body protein；the relative molecular weight of the naturally expressed gI protein was 45 ku，and the monoclonal antibody of gI protein reacted well with the gI protein expressed by intracellular infected IBRV；and by IFA，both the monoclonal antibody against gI protein and the positive control group's IBRV polyclonal antibody could specifically bind to the intracellular infected IBRV，resulting in specific green fluorescence signals in the cells，while no green fluorescence signals were found in the blank and negative control groups. In conclusion，based on the highly expressed gI protein，one cell strain gI14 that could stably express gI monoclonal antibody was successfully prepared in this study，the obtained gI protein monoclonal antibody could specifically bind to the prokaryotic expressed gI protein and IBRV whole virus，which laid a foundation for further development of any IBRV diagnostic kits as well as research on the antigen epitope and protein function of IBRV gI protein.