An acid-tolerant tyrosinase, tyrosinase-CNK, was used to form chitosan/gelatin hydrogels without any additional crosslinking materials at pH 4, where appropriate chitosan/gelatin blends were efficiently prepared based on the solubility of chitosan only under acidic conditions. The in vitro gelation degree and rheological studies revealed that the gelation of the chitosan/gelatin solution was successfully achieved within 12 min by the tyrosinase-CNK reaction at pH 4, and the glycol chitosan/gelatin blends were similarly crosslinked within 7 min at pH 6 (the optimum pH of tyrosinase-CNK). Remarkably, tyrosinase-CNK catalyzed the formation of permanent and stable chitosan/gelatin and glycol chitosan/gelatin gels, unlike the previous results of tyrosinase-mediated chitosan/gelatin gelation in which commercially available mushroom tyrosinase only catalyzed transient gel formation. In addition, the pore size distribution was greater than 100 m in the morphological structure, and the high swelling ratio and enhanced mechanical properties are expected to be suitable for tissue engineering applications. These results demonstrate that the acid-tolerant tyrosinase-CNK can efficiently mediate the in vitro formation of chitosan/gelatin conjugates, and the obtained hydrogels have the potential to be used as a promising scaffold.