Abstract: [Objective] The aim was to establish an accurate and rapid diagnostic method for detection of Brucella spp. ,Brucella abortus and Brucella melitensis. [Method] The primers and Taqman probe were designed by using BCSP31 as the specific gene and an ISTll element as the specific insertion for Brucella spp. identification. Standard positive plasmid templates were constructed respectively, and its ten-fold serial dilutions were used for making multiple quantitative PCR standard curve to evaluate the sensitivity, specificity and reproducibility of the multiplex real-time fluorescence quantitative PCR reaction system. [Result] The real-time fluorescence quantitative PCR for the identification of B. abortus and B. melitensis was established and used for the testing of clinical samples. [ Conclusion ] The establishment of the multiplex real-time fluorescence quantitative PCR diagnostic method could identify B. abortus and B. melitensis quickly and accurately.