Abstract: Streptococcus agalactiae is one of the major pathogens of tilapia (Oreochromis niloticus). At present, the virulence of strains was evaluated mainly by comparing the median lethal dose (LD₅₀) to tilapia. However, tilapia as experimental animals for evaluation of the virulence of S. agalactiae was often unstable. In this experiment, BALB/c mice were used as experimental animals, in order to establish a method for the determination of virulence of S. agalactiae. BALB/c mice were injected intraperitoneally with tilapia Streptococcus agalactiae to establish an infection model. The LD₅₀ of Streptococcus agalactiae to tilapia and mice were tested 3 times and the virulence of different Streptococcus agalactiae to tilapia and mice was determined. Results show that: by intraperitoneal injection, S. agalactiae can cause the death of mice within 24 hours. S. agalactiae could damage brain, liver, spleen, kidney and other tissues in mice. The LD₅₀ of S. agalactiae to tilapia and mice 3 times was 7.7×10⁷, 2.2×10⁸, 3.5×10⁹ CFU and 405, 361, 419 CFU, respectively. When S. agalactiae TFJ0901 and THN0901 infected tilapia (1.0×10⁷ CFU) and mice (100 CFU), the survival rates of tilapia and mouse were 100.0%, 6.7%±5.8%, and 100.0%, 0%, respectively, both of which were significantly (P<0.05) different. When S. agalactiae TFJ0901 and TFJ-F infected tilapia (3.0×10⁸ CFU) and mice (2 500 CFU), the survival rates of tilapia were 73.3%±11.5% and 80.0%±10.0%, respectively, which were not significantly (P>0.05) different from each other, while the survival rates of mice were 13.3%±11.5% and 100.0%, respectively, which were of significant (P<0.05) difference. Taken together, BALB/c mice were successfully established as a stable model for virulence determination of S. agalactiae in tilapia. The determination of the virulence of different S. agalactiae in mice is consistent with tilapia, and this model was able to distinguish S. agalactiae with similar virulence that was difficult to be distinguished by using tilapia model.