Bovine ephemeral fever is a non-contiguous epizootic arthropod viral disease infecting cattle and water buffaloes. The present work aimed to isolate the causative agent and identify it by serological characterize of the viral antigens, then molecular characterization of its genome. Infection by the bovine ephemeral fever virus (BEFV) was noticed in Dakahlyia governorates during the summer season of 2015. Twelve samples of buffy coats were collected from dairy farms suspected from clinical investigations to be infected by bovine ephemeral fever virus. The virus was isolated intracerebrally in suckling mice then successfully identified by indirect immunofluorescence technique in the brain sections of positively inoculated mice. Reverse transcriptase polymerases chain (RT-PCR) reaction was performed directly on buffy coat samples for molecular identification of the virus showed 470 bp clear band in gel electrophoresis. The sensitivity of the utilized techniques in the identification and diagnosis of bovine ephemeral fever disease revealed that the virus isolation is a gold standard technique, as well, the immunofluorescence technique. RT-PCR proved to be a rapid, sensitive and specific tool for bovine ephemeral fever disease diagnosis.