We constructed a recombinant bovine herpesvirus type 1 triple mutant virus (BoHV-1 tmv) that lacks UL49.5 residues 30-32 and 80-96, gE cytoplasmic tail (gE CT) residues 452-575 and the entire 435 bp long Us9 ORF. To develop a gE CT-specific blocking ELISA test that is necessary to distinguish the BoHV-1 tmv vaccinated calves from the wild-type (wt) virus-infected calves, a mouse monoclonal antibody (mAb) 2H8F3 was generated by using the Escherichia coli expressed gE CT residues 452-575. Further, by performing a PEPSCAN analysis of 12 mer overlapping peptides spanning the entire gE CT, the epitope sequence recognized by the mAb2H8F3 was mapped within the gE CT residues 499SDDDGPASN507. A blocking ELISA test was then developed for detecting antibodies in wild-type BoHV-1 infected calves against the gE CT epitope specified by 499SDDDGPASN507. The assay is based on the use of HRP conjugated mAb2H8F3 and the E. coli expressed gE CT protein as an indicator antibody and a coating antigen, respectively. In this assay, serum from entire gE-deleted and BoHV-1 tmv-infected calves scored negative, whereas serum from calves infected with BoHV-1 wt scored positive. Therefore, the gE CT-ELISA, based on the mAb2H8F3 and E. coli expressed gE CT protein, is suitable for differentiating the wt virus-infected and BoHV-1 tmv-vaccinated cattle.