Chitosan/gelatin and chitosan/gelatin/nanohydroxyapatite hydrogels were rapidly and stably prepared without any crosslinking materials by using an engineered tyrosinase (mTyr-CNK) with high catalytic activity for tyrosine/DOPA-tethered polymeric biomaterials throughout a broad pH range. A dual-barrel syringe with one part containing chitosan/mTyr-CNK solution and the other containing gelatin solution with/without nanohydroxyapatite was successfully used to form homogeneous hydrogels at room temperature followed by 37 oC to simulate an in situ injection approach. The obtained hydrogels exhibited an average pore size greater than 150 m and high swelling ratios with similar mechanical properties to other chemically crosslinked chitosan/gelatin hydrogels. The in vitro degradation properties and cellular viability suggested that the hydrogels could be used as biodegradable and biocompatible scaffolds for biomedical applications, such as space filling biomaterials and delivery vehicles for bioactive molecules and cells. These results demonstrated that mTyr-CNK-mediated hydrogels have remarkable promise as an injectable scaffold biomaterial.