In this study, bovine herpesvirus 1 gE deleted vaccine was prepared from local isolate, by replacing entire gE coding region with enhanced green fluorescent protein (EGFP) for selection. After transfection of MDBK cells with genomic viral DNA and EGFP-bearing gE-deletion plasmid, fluorescent recombinant clones were produced. Deletion of gE gene was confirmed by PCR. BoHV-1ΔgE recombinants formed smaller plaques but replicated in similar titer and kinetics as compared to parental virus (UVAS/2) on MDBK cell lines. In-vivo study showed that locally prepared BoHV-1ΔgE vaccine induced good immune response. For confirmation of seroconversion of gE, serum samples were also tested by gE competition ELISA. The results showed that all rabbits vaccinated with recombinant BoHV-1ΔgE remained negative in gE competition ELISA. The study concluded that locally prepared BoHV-1ΔgE marker vaccine can be candidate vaccine to prevent BoHV-1 infections in Pakistan.