Streptococcus agalactiae is a causative agent of streptococcosis disease in various fish species, including Nile tilapia (Oreochromis niloticus Linn.). Vaccination is an effective disease prevention and control method, but limitations remain for protecting against catastrophic mortality of fish infected with different strains of streptococci. Immunoproteomics analysis of S. agalactiae was used to identify antigenic proteins and construct a chimeric multiepitope vaccine. Epitopes from five antigenic proteins were shuffled in five helices of a flavodoxin backbone, and in silico analysis predicted a suitable RNA and protein structure for protein expression. 45F2 and 42E2 were identified as the best candidates for a chimeric multiepitope vaccine. Recombinant plasmids were constructed to produce a recombinant protein vaccine and DNA vaccine system. Overexpressed proteins were determined to be 30 kDa and 25 kDa in the E. coli and TK1 systems, respectively. The efficacy of the chimeric multiepitope construct as a recombinant protein vaccine and DNA vaccine was evaluated in Nile tilapia, followed by S. agalactiae challenge at 1 × 107 CFU/mL. Relative percentage survival (RPS) and cumulative mortality were recorded at approximately 57-76% and 17-30%, respectively. These chimeric multiepitope vaccines should be applied in streptococcosis disease control and developed into a multivalent vaccine to control multiple diseases.