In this study, the specific polymerase chain reaction has been standardized and evaluated for the direct diagnosis of Brucella canis in vaginal swab samples from dogs. The specific primer sets are directed to the 16S-23S rRNA inter-space region of Brucella spp. and the deletion of 351 bp in BMEI1426-BMEI1427 in B. canis. A total of 21 references and field strains and 35 vaginal swab samples were used for the evaluation of the polymerase chain reaction. It found that polymerase chain reaction is positive for B. canis DNA indicated by only amplification of 214 bp product. It detected at least 2.7 × 101 CFU/g of bacteria diluted in vaginal swab samples indicates that the polymerase chain reaction can be used as a practical alternative for bacterial isolation. The novel polymerase chain reaction provides a simple and rapid for the detection of B. canis in clinical and field samples in one step and in short time about 24 h.