In teleost, cathepsin B has been identified from several species and shown to play roles in the host immune response during pathogen challenge. However, the mechanism of how cathepsin B modulates the immune response in teleosts remains poorly understood. In this study, we identified and characterized cathepsin B (LycCatB) and invariant chain (LycIi) from the large yellow croaker (Pseudosciaena crocea). Sequence comparison and phylogenetic analysis indicated that LycCatB and LycIi are highly conserved within teleosts. Quantitative RT-PCR analysis showed that LycCatB mRNA was widely expressed in all examined tissues. We then recombinantly expressed LycCatB and Lyc-TR-Ii (transmembrane domain removed Ii chain) in Pichia pastoris and Escherichia coli, respectively. The recombinant LycCatB (rLycCatB) can hydrolyze the substrate Z-FR-AMC with a Km value of 40.68μM. Furthermore, co-incubation of rLycCatB with rLyc-TR-Ii led to an efficient cleavage of rLyc-TR-Ii in a time-dependant manner. These results indicated that cathepsin B may be involved in MHC class II-associated Ii processing in large yellow croaker, and provide new information helping to elucidate the immunological functions of teleost cathepsin B.