The study was applied on 4772 lactating and non-lactating cows distributed on different districts in Al Sharqia Governorate. Sera were collected from animals during routine diagnosis and control program. The results of screening tests Buffer acidified plate antigen test (BAPAT), Rose Bengal plate test (RBPT), Tube agglutination test (TAT), Complement fixation test (CFT) and indirect enzyme linked immunosorbent assay gave 124 and 176 seroreactive animals by incidence of 4.42% and 8.91% in private farms and individual cases respectively. 37 (29.8%) and 97 (55.1%) isolates of Brucella melitensis biovar 3 were recovered from 124 and 176 seroreactive animals respectively. In seroreactive cows, Brucella melitensis biovar 3 was isolated from 36% and PCR yielded expected products in 40%. In conclusion, more attention should be paid to the role of Brucella melitensis biovar 3 in brucellosis in cattle during the application of national program of brucella control and eradication.