In order to detect the antigenicity of moonlighting protein EF-Tu (Elongation Factor Tu), EF-Tu gene from Streptococcus agalactiae HN0303 isolated from tilapia was cloned. The related properties of EF-Tu protein were predicted and its phylogenetic tree was also constructed. The rEF-Tu protein was obtained by prokaryotic expression systems and purified by Ni-NTA-Sefinose Column. The purified rEF-Tu protein immunized rabbits were used to obtain the polyclonal rabbit anti-rEF-Tu sera for antigenicity detection. The results showed that EF-Tu gene had an ORF with 1197 bases, encoding 398 amino acids with a C₁₉₃₃H₃₀₉₆N₅₃₂O₆₁₅S₁₁ formula, 43.981 ku molecular mass, and a 4.749 theoretical isoelectric point. Futhermore, the deduced amino acids comprised phosphorylation sites, but did not contain the transmembrane domain and signal peptide sequence. Three conserved domains, namely EF-Tu, EF-Tu-II and EF-Tu-III existed in the deduced amino acids via NCBI Conseverd Domains tool. Phylogenetic analysis revealed an exaggerated degree of homology with other S.agalactiae EF-Tu protein. Additionally, high antigen index of the deduced amino acids was predicted using DNAstar-Protean, which means it can form numerous epitopes. rEF-Tu proteins formed into inclusion bodies were found in the pellet and an about 66.4 ku band was observed by SDS-PAGE. Moreover, Western Blot analysis showed that rabbit anti-rEF-Tu sera can combine the tropina with recombinant protein specifically. Adhesion test suggested that rabbit anti-rEF-Tu sera prevented S. agalactiae HN0303 adhering the EPC(Epithelioma papulosum cyprini) with a decrease of 79.99%±2.43%. In this study, our results showed that rEF-Tu possesses nice antigenicity and the rabbit anti-rEF-Tu sera can inhibit the S. agalactiae HN0303 adhesion obviously, which suggested that the EF-Tu protein may become a subunit vaccine candidate against S. agalactiae in tilapia.