This study aimed to detect Brucella antibodies in the sera of dairy cows and to identify Brucella species in the milk of seropositive cows. A total of 100 sera and 100 milk samples were collected from two 50-cows groups (group 1 with and group 2 without a history of reproductive problems and/or decreased milk production). Rose Bengal plate test and indirect ELISA were used to explore Brucella antibodies in the serum samples and thereafter milk samples of seropositive cows were undergone PCR analysis using Brucella genus specific primers and 3 pairs of species specific primers for identification of B. abortus, B. melitensis and B. suis. The RBPT showed 22 cows were carriers for the Brucella antibodies, 18 in group 1 and 4 in group 2 whereas the iELISA showed only 10 cows out of these 22 cows were positive, 9 in group 1 and only 1 cow in group 2. The PCR assay, which was performed on milk samples of the RBPT positive cows, revealed 18 samples were positive for the Brucella genus and the Brucella abortus species and were negative for Brucella melitensis and Brucella suis species. As a conclusion, the results of this study showed that brucellosis has been encountered in cows with or without a history of reproductive problems, and the RBPT followed by PCR assay for milk samples of the seropositive cows could provide more specific detection than performing either test alone and could be more useful for rapid screening of brucellosis in dairy cows.