In order to identify Nocardia quickly and accurately, three primers were designed for detection of rpoB, SecA1 and 16S rRNA genes. The specificity of simplex polymerase chain reaction (PCR) was verified. Then multiple PCR method was established, and the sensitivity and specificity were tested by 44 Nocardia standard strains, 44 clinical isolates and 7 reference strains under the same reaction system and condition. The results showes that 2 strains of Nocardia (DSM 43003 and CDC 51) were amplified by using a single primer pair, and the single bands obtained were consistent with target fragment. Then the target genes were verified by sequencing and Basic Local Alignment Search Tool (BLAST). With the established multiple PCR method, rpoB, SecA1 and 16S rRNA segments were confirmed in 43 (97.7%) of 44 Nocardia standard strains and 42 (95.5%) of 44 clinical isolates, however, there were no bands obtained in 7 reference strains. The specificity met the test requirement, and the detection limit for DNA template was 1×10^-4 ng. It is concluded that multiple PCR method is fast, accurate, specific and sensitive. It can be used for identifying Nocardia strains.