During 2000-2001 outbreaks of bovine ephemeral fever (BEF) occurred at Middle Egypt, 251 heparinized blood samples from cattle and buffaloes with BEF symptoms were collected for virus isolation and identification and 549 serum samples were collected for serological examination. Suckling baby mice were intracerebrally (I/C) inoculation for virus isolation and identification, in the same time isolated virus was adapted to grow in both Verol₂₁ and BKH₂₁ cell line. Identification of isolated virus was achieved using both reference and prepared hyper immune serum against BEF virus in rabbit by using SPA agglutination test (SPA), Serum neutralization test (SNT), Mouse protection Test (MPT) and Agar gel precipitation test (AGPT).