Adenylate kinase (ADK), which could be applied in adenosine triphosphate (ATP) regeneration, reversibly catalyzes the conversion of two adenosine diphosphate (ADP) molecules to ATP and adenosine mo-nophosphate (AMP). As free enzymes have the narrow range of reaction temperature and pH adaptation, poor stability and non-reusability, they are expensive in application and not suitable for industrial-scale produc-tion. In order to achieve the immobilization of ADK, the expression plasmid pET-ADK was first constructed, and recombinant ADK was obtained by isopropylthio-β-D-galactoside (IPTG) induction of E. coli BL21(DE3). Then, the chitosan concentration for 3D bioprinting, the pH value of enzyme solution, enzyme concentration and immobilization time in the process of immobilization were optimized. Finally, the physicochemical proper-ties and ATP regeneration ability of 3D-bioprinted immobilized ADK were examined. The results showed that the optimal conditions for ADK immobilization by 3D bioprinting using chitosan-based bioink were 3% of chitosan concentration, pH 7.5, 0.10 mg/mL of enzyme concentration, and 3 h of immobilization time. By using 3D-bioprinted immobilized ADK hydrogels, the yield of D-glucose-6-phosphate increased by 52.00% when glucose was catalyzed with glucose kinase. In addition, after repeated use for 8 times, the immobilized ADK still retained 74.83% of its initial activity. These results provide the groundwork for studying ADK im-mobilization that can be applied to ATP regeneration.