Brucellosis is endemic in Egypt, so wherever herd problem associated abortion is present, brucellosis should be suspected, and its sero-diagnosis is needed. This study aimed to determine the seroprevalence of brucellosis in Different Governorates in Egypt and isolation and biotyping of Brucella isolated from Egypt confirmed by PCR. A total of 1857 samples were collected including 1531 serum, 148 milk, 58 lymph nodes,58 spleen samples, 58 liver samples and 4 aborted foeti from cattle in 7 Governorates in Egypt. Serological tests; Rose Bengal Plate Test (RBPT), Buffered acidified plate antigen (BAPA) test, modified standard tube agglutination (MSTA) and indirect ELISA were applied on positive serum samples for (RBPT). Brucella was isolated and identified from milk, lymph nodes, Spleen and aborted foeti. The results detected 19 isolates from (aborted foeti 1, milk 8, lymph nodes 8 and spleen 2) were detected and identified as B. melitensis biovar3. The results of RBPT, BAPA, MSTA and indirect ELISA tests were 21.8%, 23.7%, 80.2%, and 89.8% respectively. MSTA and indirect ELISA applied on positive sera of RBPT. Multiplex PCR was applied as a confirmation and rapid detection of B. melitensis isolates. all isolates showed positive results with oligonucleotide primer that amplified a 731bp fragment confirmed as B. melitensis. In conclusion, Serology remains the most practicable method for diagnosis of brucellosis, no currently available single serological test can be considered reliable for the detection of brucellosis and the gold standard for diagnosis of brucellosis is the isolation and phenotypic characterization of the organism. A combination of growth characteristics, serological, bacteriological or molecular methods is required for a definitive identification.