Abstract: By designing primers and probe based on the conserved region of cfb gene of Streptococcus agalactiae isolated from tilapia, we developed a real-time fluorescent PCR assay for detection of S.agalactiae from tilapia tissue andverifiedthe specificity, sensitivity and practicability of the assay. The standard and positive strains of S.agalactiae were positive, and all negative controls were negative. The minimum detectable concentration of S.agalactiae genome DNA was 3.4210-7 ngL-1, and the detection limit of the assay was less than 10 cells per reaction system. The genome DNA samples of midgut, liver, spleen and kidney which were collected from tilapia artificially infected with S.agalactiae were tested by real-time PCR and were positive. The quantities of S.agalactiae detected in per g DNA of tissue samples were 2.95103 cells, 2.45107 cells, 2.34103 cells and 4.54104 cells, respectively, and the controls were negative. The results show that the real-time quantitative PCR assay which we developed was accurate, specific and sensitive, and could detect S.agalactiae from various tilapia tissues rapidly. The method can be used for surveillance and prevention of S.agalactiae disease of tilapia.