Abstract: Two pairs of specific primers were designed and synthesized according to the 16SrRNA and Surface immunogenic protein(sip)gene sequence of tilapia Stretococcus agalactiae,and a double-PCR method for rapid detection of Streptococcus agalactiae isolated from tilapia was established through optimization of reaction system and reaction condition,by which two specific fragments for S.agalactiae of 1 305bp and 121bp were producted. Specificity test showed that two specific bands,16SrRNA and sip,could only be detected in the S.agalactiae, and none was amplified from Streptococcus inia.The minimum detectable quantity of the S.agalactiae strains 070717LL was 1.05×102 CFU.In the tissues from spleen,brain and kidney of tilapias affected by the S.agalactiae strain 070717LL the strains DNA were detected,especially in kidney and spleen tissues.In conclusion,this double-PCR method was specific,sensitive rapid,and applicable for epidemiological investigation of S.agalactiaein tilapia.