The aim of the present study was to determine comparatively the presence of anti-Brucella antibody and Brucella DNA in cow milk. Anti-Brucella antibody was detected by ELISA based on the lipopolysaccharide (LPS) as diagnostic antigen. Besides, the presence of Brucella DNA in milk samples was screened by eryCD gene-targeted PCR and B. abortus DNA was determined by amplification of alkB genes. For this purpose, 70 raw cow milk samples collected from open markets were used. Among these samples, 15 samples (21.4%) were found positive for anti-Brucella LPS antibody in ELISA. In contrast, only 5 milk samples (7.1%) were determined as positive by eryCD gene-targeted PCR. All of the eryCD positive samples giving an amplicon of 904 bp indicated the presence of wild-type Brucella DNA but not B. abortus S19 vaccine strain allowing amplification of only an amplicon of 202 bp. In addition, amplification of the alkB gene demonstrated the presence of B. abortus DNA in 5 eryCD positive samples. No statistical agreement was observed between ELISA and PCR results with 95% confidence interval. These results strongly suggest that use of both ELISA and PCR methods could lead to more reliable diagnosis of brucellosis from bovine milk samples.