Abstract: To investigate the expression and function of Trachinotus ovatus cathepsin B (TroCatB) gene in response to bacterial stimulation, TroCatB cDNA was cloned by RT-PCR and RACE technology and it was 2181 bp in length with a 391 bp 5′UTR, a 797 bp 3′UTR and an 993 bp ORF encoding of 330 amino acid residues. The estimated molecular mass and theoretical isoelectric point were 36.37 kD and 5.73, respectively. The TroCatB protein has a signal peptide (¹Met-¹⁸Ala), a precursor peptide (²⁵Leu-⁶⁴Gly) and a typical papain family cysteine domain, containing ¹⁰⁷Cys, ²⁷⁷His, ²⁹⁷Asn catalytic activity sites. Homology analysis showed that the homology of the TroCatB protein with other vertebrates was 67.0%—90.9%, and the homology of the mature peptide region with other vertebrates was 73.7%—92.4%. The NJ phylogenetic tree revealed that the scorpionfish and other fish clustered together, closest to the corpus callosum. Real-time quantitative PCR indicated that TroCatB mRNA expressed in various tissues with the highest level in spleen. Vibrio alginolyticus infection significantly induced the expression of TroCatB gene in spleen with peak level at 6h and head kidney tissues (P<0.05) with peak level at 12h. These results indicated that the domain and catalytic active sites of TroCatB protein are conserved during genetic evolution. The TroCatB gene regulates the physiological activities of the organism against bacterial immunity, and plays an important role in the innate immune defense of ovate to elucidate the function of TroCatB in the immune process and the pathogenesis of pathogens.