In this study, 482 serum and tissues samples including spleen and lymph nodes were collected from cattle (215), buffaloes (103), sheep (105) and goats (59), which had been slaughtered out of abattoirs in different localities in Assiut Governorate. The serum samples were examined for the detection of antibodies against Brucella spp. The results of screening tests Buffer acidified plate antigen test (BAPAT) and Rose Bengal plate test (RBPT) gave 36 seroreactive animals by incidence of 10.23% in cattle, 2.91% in buffaloes, 7.61% in sheep and 5.08% in goats, respectively. All positive serum samples were further retested by Standard serum agglutination test (SAT), Rivanol test (RIVT) and indirect enzyme linked immunosorbent assay (ELISA) as confirmatory tests. SAT gave 90.9% in cattle and 100% in buffaloes while RIVT gave 86.3% in cattle and 100% in goats of serorective animals. Moreover, ELISA gave 95.4% in cattle, 100% in buffaloes, 87.5% in sheep and 100% in goats of the seroreactive animals. Eleven isolates (30.3%) of brucella melitensis biovar 3 were recovered from 36 seroreactive animals. These isolates represent 6 (27.3%) for cattle, 1 (33.3%) for buffaloes, 3(37.5%) for sheep and 1(33.3%) for goats. In conclusion, Brucella melitensis was wide spread in ruminants slaughtered out of abattoirs in Assiut Governorate which cause a serious infection in human and animals.