In order to detect the antigenicity of moonlighting protein FBA (fructose-1,6-bisphosphate aldolases), fba gene from Streptococcus iniae DX09 isolated from Ictalurus punctatus was cloned. The related properties of FBA protein were predicted and its immunization efficacy assay was conducted. rFBA protein was obtained by prokaryotic expression systems and purified by Ni-NTA-Sefinose Column. The purified rFBA protein was used to immunize rabbits (Oryctolagus cuniculus) to obtain the polyclonal rabbit anti-rFBA sera for antigenicity detection. The results showed that fba gene had an ORF with 882 bases, encoding 293 amino acids with a C₁₃₇₈H₂₁₇₂N₃₆₈O₄₂₂S₈ formula, 30.9 ku molecular mass, and a 5.01 theoretical isoelectric point. Furthermore, the deduced amino acids comprised phosphorylation sites, not containing the transmembrane domain and signal peptide sequence. The conserved domains, namely aldolase were predicted via NCBI conseverd domains tool. The comparative analysis revealed an exaggerated degree of homology with other S. iniae FBA protein in amino sequences. Additionally, high antigen index of the deduced amino acids was predicted using DNAstar-Protean, which means it can form numerous epitopes. rFBA proteins formed into inclusion bodies were found in the pellet and a band about 47 ku was observed by SDS-PAGE. Moreover, western blot analysis showed that rabbit anti-rFBA sera can combine with the mycoprotein specifically. Immunization efficacy assay suggested that rFBA prevented S. iniae DX09 infecting I. punctatus with a 55% protective rate. In this study, our results showed that rFBA possesses nice antigenicity and optimal immune protection, implying that the FBA protein can be a subunit vaccine candidate against S. iniae in I. punctatus.