In recent years, Streptococcus iniae infected farmed tilapia causes economic losses in our country and the world. To set up a method detecting S. iniae in tilapia, this paper designed primers CM1/CM2 based on the sequences of S. iniae. At the same time, the tests which amplified the specific DNA fragment, optimized the PCR reaction condition, sensitiveness and special were detected. The different detected material was compared and 9 clinical samples were detected at one time. The result indicated that the CM1/CM2 primers set only amplified a specific DNA fragment from S. iniae but not from 9 strains common pathology bacteria of fisheries. It can detect bacterial cells in 20 -30, and the PCR reaction also can directly detect the S. iniae from brain, liver, kidney, spleen of infected tilapia. On the other hand, the detection results of clinical strains were consistent with the Phylogenist Analysis based on 16S rRNA gene sequences. The method makes up for the disadvantage of traditional method that can not detect the species between the bacteria and reduce the detected time and the cost. So, the method detecting S. iniae will have a good future for application.