Bluetongue disease (BT) is a notifiable disease affecting ruminants causing devastating economic losses globally. In the current study, the prevalence of Bluetongue virus (BTV) antibodies in sera  of clinically infected Baladi and Merino sheep, goats, and cattle, from South Sinai areas and Monib abattoir (during Eid El-Adha), were investigated by competitive ELISA. Seropositivity of goats and Baladi sheep sera were 77.8% and 46.3% denoting to the highest prevalence of BT virus in these animals, but sera of Merino sheep and cattle were negative denoting to the freedom of this lot of imported animals from previous vaccination or infection. BTV was isolated on SPF-ECE via yolk sac route yielding hemorrhagic embryos, also on (CAM) with the appearance of pock lesion, but secondary isolation on (BHK-21) demonstrated cytopathic effects (CPE) typical of viral infection. Concentration of BTV was performed using PEG-6000. BTV and aggregation of virus particles were observed with specific BTV antibodies and photographed by electron microscopy (EM). Titration studies (TCID50) was approximately 10-7 TCID50/ml. Finally, the identification of Bluetongue virus was accomplished and confirmed molecularly using shotgun proteomic analysis demonstrating the presence of VP1 protein during year 2016 in Egypt.