[Objectives] This study aimed to clone the tyeA gene of Vibrio alginolyticus HY9901 strain and analyze its sequence by bioinformatics. [Methods] By referring to the entire genome sequence of V. alginolyticus on GenBank, specific primers were designed. According to the principle of PCR amplification, the target gene tyeA was amplified. By means of bioinformatics, the sequence of tyeA was further analyzed, and the phylogenetic tree of tyeA genes of Vibrio spp. and the corresponding subunit three-dimensional structure models were constructed. [Results] The length of the tyeA gene of V. alginolyticus strain HY9901 is 285 bp, and its theoretical molecular weight is 10.98 kD. According to prediction, there is no signal peptide or transmembrane region at the N-terminus of the sequence, and the amino acid sequence contains two casein kinase II phosphorylation sites. The results of protein subcellular localization prediction show that the TyeA protein is located in the cell membrane. The protein is unstable and non-hydrophilic. The tertiary structure of TyeA protein of V. alginolyticus is similar to that of Yersinia sp. According to prediction, TyeA has a major functional domain Pfam. In terms of secondary structure, alpha helix, random coil, extended strand and beta turn account for 85.11%, 7.45%, 4.26% and 3.19%, respectively. The homology of Tye A between V. alginolyticus and Vibrio parahaemolyticus is up to 83%, so they are classified into one cluster. [Conclusions]This study will help to further understand the regulation mechanism of type III secretion system in V. alginolyticus.