Zeolitic imidazole frameworks (ZIFs) with tunable pore sizes and large surface areas have been widely used as effective supports for enzyme/cell immobilization. In this study, a bi-enzyme system (BP@ZIF-8) was developed for the biosynthesis of Ala-Gln coupled with efficient ATP regeneration. BP@ZIF-8 was then successfully fabricated and characterized. Results showed that BP@ZIF-8 exhibited high thermostability at 50 °C and high pH stability in neutral and alkaline reaction buffers. However, BP@ZIF-8 displayed poor pH stability in acidic environments. In addition, it could maintain 58.9% of their initial activity after 7 continuous uses, demonstrating satisfactory reusability. When applied for the batch production of Ala-Gln, the yield of Ala-Gln reached to about 14.56 mM after 14 h, an increase of approximately 14.3%. These demonstrated that BP@ZIF-8 shows great potential for the practical biosynthesis of Ala-Gln. [ABSTRACT FROM AUTHOR]