Abstract: The gene, which codes the amid acid residency from 192th to 266th of Infectious Bovine Rhinotracheitis Virus(IBRV) glycoprotein B(gB), was amplified by PCR technique with specific primers after analyzing glycoprotein B with biosoftware DNAstar. Then the amplified product was cloned into Tvector. After identifying with doubleenzyme cut and sequencing for the accuracy of the product of amplification, the expected band on the agarose gals was recovered and directional inserted into pET32a vector. The recombinant vector was transformed into BL21. SDS-PAGE analysis showed that the fusion protein of 29.2 ku as expected was in solution forms partially. The recombinant protein was purified by affinity chromatography, the concentration of purified protein was 2.000 mg/mL and the purity was 79.2%. The recombinant protein was checked by the indirect enzymelink assay, which showed that it retains antigenicity of the nature protein. An indirect enzyme linkedassay(antigen concentration: 50 μg/mL; serum dilution: 1∶40) was developed with the recombinant protein as a diagnosis antigen, the positive criterion was S/P>0.395There was no cross reaction with positive sura of other six diseases, including BEF(Bovine ephemeral fever), CBPP(Contagious bovine pleuropneumonia), BVD-MD(Bovine viral diarrheamucosal disease), BTB(Bovine tuberculosis), BPB(Bovine paratuberculosis) and BAD(Bovine Akabane disese). Comparing with the Virus Neutralization testing, the speciality was 83.3%; the sensitivity was 90.9% ;the accordance rate was 88.9%. Comparing with the IBR ELISA kit from France, the speciality was 92%; the sensitivity was 92.7% ; the accordance rate was 92.5%. The assay was confirmed to be specific and sensitive by the results above, suggesting a good application prospect.