[Objectives] To clone and analyze the TpiA gene of Vibrio alginolyticus HY9901. [Methods] According to the TpiA gene sequence of V. alginolyticus, a pair of specific primers was designed, and its full length was amplified by PCR. [Results] The full length of TpiA gene is 771 bp, encoding 256 amino acid residues in total, and the NCBI accession number is OM906798. According to the deduced amino acid sequence, its molecular weight was predicted to be about 26.975 48 kDa, and its isoelectric point was 4.78. The amino acid sequence of the N-terminal signal peptide structure was predicted, and it was found that there was no obvious signal peptide cleavage site, no signal peptide, and no transmembrane region; the amino acid sequence contained 3 N-glycosylation sites, 4 protein kinase C phosphorylation sites, 2 casein kinase II phosphorylation sites, 6 N-myristoylation sites, 7 microbody C - terminal target signal site, and 1 triose phosphate isomerase active site. The prediction results of protein subcellular localization showed that TpiA may be located in mitochondria or cytoplasm, with probability of 39.1% and 34.8%, respectively. The amino acid sequence of the TpiA gene of V. alginolyticus shared 98.83%-99.61% homology with other Vibrio species, and it was clustered into the same subfamily with Vibrio parahaemolyticus and had a close relationship. In the secondary structure prediction, the proportions of α-helix, random coil and extended chain were 44.53%, 41.41% and 14.06%, respectively, and the similarity of its tertiary structure model to template 1aw1.1.A was 85.16%. [Conclusions] This study is intended to provide a basis for further research on the role of TpiA gene in the type III secretion system and related research on antibiotic resistance.