Abstract: Nile tilapia (Oreochromis niloticus) is an important commercial fish in aquaculture in the world. However, it is easy to be infected by Streptococcus iniae in the breeding production. The use of vaccine is a relatively ideal anti infection measure. In this study, eukaryotic expression vectors (DNA vaccine) were constructed by taking the simA and pgmA genes of S. iniae as the target genes. Then protective efficacy was evaluated by intramuscular injection of tilapia. Two target genes were detected in the injected fish at DNA and RNA levels after immunization. Besides, 7−28 d  after the first immunization, the expression levels of interleukin-1 (IL-1β) and Tumor Necrosis Factor (TNF-α) in the gill, liver, kidney and head kidney of the vaccinal group were higher to some extent than those in the PBS control group. The antibody titer and antibacterial activity of immunized groups were significantly higher than those of control groups (P<0.05). Two weeks after being challenged with S. iniae, the relative percent survival of mixed vaccines of pcDNA3.1-pgmA, pcDNA3.1-simA and pcDNA3.1-pgmA, with pcDNA3.1-simA were 60.7%, 49.9% and 75.0%, respectively. It is revealed that the vaccine prepared in this paper has immune protection effect and can be used as a candidate vaccine.