The mucosal surfaces of fish (skin, gill and intestine) constitute the primary line of defense against pathogen invasion. Although the importance of fish mucosal surfaces as the first barriers against pathogens cannot be overstated, the knowledge of teleost mucosal immunity are still limited. Cathepsin B, a lysosomal cysteine protease, is involved in multiple levels of physiological and biological processes, and playing crucial roles for host immune defense against pathogen infection. In this regard, we identified the cathepsin B (ctsba) of channel catfish and investigated the expression patterns of the ctsba in mucosal tissues following Edwardsiella ictaluri and Flavobacterium columnare challenge. Here, catfish ctsba gene was widely expressed in all examined tissues with the lowest expression level in muscle, and the highest expression level in trunk kidney, followed by spleen, gill, head kidney, intestine, liver and skin. In addition, the phylogenetic analysis showed the catfish ctsba had the strongest relationship to zebrafish. Moreover, the ctsba showed a general trend of up-regulated in mucosal tissues following both Gram-negative bacterial challenge. Taken together, the increased expression of ctsba in mucosal surfaces indicated the protective function of ctsba against bacterial infection, and the requirement for effective clearance of invading bacteria. Further studies are needed, indeed, to expand functional characterization and examine whether ctsba may play additional physiological and biological roles in catfish mucosal tissues.