ObjectiveTo establish multiplex PCR assay for detects Brucellosis in laboratory canine and related biological products． Method These primers of multiplex PCR were designed according to the Omp2 gene of Brucellosis，and the PCR reaction was optimum，and then some samples were digested by three restriction enzyme for differentiate Brucellas． We also cloned PCR products and measured sequence after multiplex PCR to determine the accuracy of the assay． Then the specificity and sensitivity of the assay was tested． Results The target bands were successfully amplified，and these samples were successfully distinguished by digestion of the PCR product; It was 99% homology between the target segment by multiplex PCR and Brucellosis DNA sequence from GeneBank． The specificity of this method is better，1. 8 × 10 － 7 μg /ml samples can be detect． Conclusion The Brucella PCR detection and typing of multiple identification methods was established successful，the assay have good specificity and high sensitivity． The assay is important to ensure the quality of laboratory canine and protect the health of the animal keepers and laboratory personnels．