Bovine herpesvirus-1 (BoHV-1) is responsible for different clinical findings in cattle including tracheitis, conjunctivitis, keratoconjunctivitis, vulvovaginitis, balanoposthitis, abortion, infertility, encephalitis and fatal disease in newly born calves, and causes high economic losses. The present study was carried out for diagnosis of BoHV-1from clinically suspected cattle in El Menoufiya governorate, Egypt during the period from September 2021 until December 2021. A total of 50 field samples including swabs (n= 35) and internal organs (n= 15) collected from clinically suspected cattle were isolated on cell culture of Madian Darby Bovine Kidney (MDBK) cell line. The isolates were identified using Antigen detection Enzyme Linked Immunosorbent Assay (ELISA) and conventional Polymerase Chain Reaction (PCR). Out of 50 tested samples, 30 samples Produce CPE on MDBK cells by third passage. The ELISA only identified BoHV-1 in 20 tissue culture isolates and confirmed by the PCR amplicon of 575 bp targeting Glycoprotein C gene. it was concluded that, isolation, antigen detection ELISA and conventional PCR based on the Glycoprotein C (gC) were efficient for the identification of BoHV-1virus. This indicates circulation of BoHV-1 on Menoufiya governorate and directed toward application of control measures.