Brucellosis is one of the most prevalent bacterial zoonosis worldwide. Brucella melitensis (B. melitensis), Brucella abortus (B. abortus), Brucella canis (B. canis), Brucella suis (B. suis) are common to cause disease in humans and B. melitensis is the most pathogenic causative agent of brucellosis in humans and animals. Fast, efficient and accurate identification of Brucella reference strains at the strain-level is indispensable for microbiological method quality assurance and downstream applications. B. melitensis 63/9 is recognized as an important reference strain for the microbiological culture collection organizations worldwide, and the identification of B. melitensis strain 63/9 is still lacking. The genomic sequences of B. melitensis 63/9 and nine other Brucella strains were compared. Two specific genes were selected for the multiplex PCR method. Gene BMEA_B0162 with unknown function is the key target to identify B. melitensis 63/9, and gene BMEA_A1238 annotated as TRAP transporter solute receptor is included as a control gene for the Brucella genus. A multiplex PCR was established in this study to differentiate B. melitensis reference strain 63/9 from 39 B. melitensis strains, 13 B. abortus strains, 5 B. suis strains, 6 B. canis strains, 3 E. coli, and 4 Salmonella strains by targeting the BMEA_B0162 and the BMEA_A1238 in the genome. This method allows at least 100 pg of B. melitensis 63/9 genomic DNA to be detected. We established a fast, and a cost-effective method to distinguish B. melitensis 63/9 from other Brucella strains and some non-Brucella bacteria strains with high sensitivity and specificity, making the first report about the identification of Brucella reference strain recognized by World Organization for Animal Health at the strain-level.