Enolase (ENO) is one of the surface-exposed proteins of Streptococcus iniae, which previously had been identified as a plasminogen-binding protein. In this study, ENO was evaluated to induce cross-protective immunity against S. iniae and Streptococcus parauberis which are major pathogens causing streptococcosis in fish. Immunoblot analysis shows that S. iniae recombinant ENO (rENO) produced in Escherichia coli was cross-reactive with antisera against S. iniae, and S. parauberis serotype land II. In the challenge experiment of streptococcal infection after vaccination in zebrafish, rENO elicited a similar protection with a whole cell bacterin against S. iniae and S. parauberis, which suggests its feasibility as an efficient vaccine against streptococcosis.