To study the immune efficacy of inactivated vaccine of heterologous Streptococcus agalactiae extracellular product in tilapia (Oreochromis niloticus), two strains of S. agalactiae (HN0901 and GX1101) were cultured, and highly efficacious vaccines were developed using extracellular products and formalin-killed whole cells of HN0901 and GX1101. The vaccine was administered by intraperitoneal (i.p.) injection in a volume of 0.2 mL, and sham-vaccinated (control) fish were i.p. injected with the same volume of sterile BHI. At 28 d post-vaccination (DPV), the fish were challenged, i.p. injection, with approximately 1.0×108 CFU of homologous or heterologous S. agalactiae to determine the efficacy of protective immunity. The immune response and serum antibody titer of the immunized and post-injected tilapia were measured, and the relative immune protection rate and cross-immune protection rate were compared. The results showed that the alanine aminotransferase and aspartate aminotransferase levels of immunized fish were significantly higher than those of control fish at 28 DPV but significantly lower than those of control fish post injection. Compared with the control fish, the levels of superoxide dismutase and catalase in immunized fish increased in varying degrees after immunization and challenge, while malondialdehyde levels decreased significantly (P＜0.05). The results of real-time quantitative polymerase chain reaction also indicated that TNF-α, IL-1β, TGF-β, HSP 70, MHC , Ⅱ and IgM expression levels showed different degrees of significant increase and were higher than those in the control group after immunization and challenge (P＜0.05). Serum antibody titer reached 1 : 6400 at 28 DPV, which was significantly higher than that of the control group (P＜0.05). The relative percentage of survival ranged from 31.6% to 47.4% after the homologous or heterologous S. agalactiae challenge. Furthermore, the developed inactivated vaccines significantly induced an immune response in tilapia and produced moderate vaccine efficacy. Administration of the single isolate HN0901 or GX1101 vaccine via i.p. injection was marginally protective in preventing streptococcal disease caused by homologous or heterologous isolates of S. agalactiae. Our findings laid a foundation for further studies on immune prevention technology against streptococcosis in tilapia and the development of bivalent vaccines.