<p>Leveraging metal-organic frameworks (MOFs) to encapsulate fragile enzymes presents an advanced strategy for creating robust biocatalysts. This method substantially enhances the stability of enzymes but usually compromises its catalytic activity as the mass transfer limitation imposed by the MOF structure. Herein, we report a conceptual enzyme encapsulation scheme using a water-impregnated microporous MOF, demonstrating the positive role of the water clusters inside the MOF micropores in facilitating enzymatic reaction. We discover that the water clusters not only promote the hydrophilicity of the pore channel microenvironment but also mitigate undesirable interactions between substrate molecules and the pore walls via an unusual steric effect, which can otherwise block substrate diffusion and hinder mass transfer. The activation principle on enzymatic reaction herein is completely different from the reported strategies relied on pore-defect engineering, which encompasses complex synthesis steps and lacks flexibility in precise structure regulation. Furthermore, we demonstrate the potential of this waterimpregnated MOF biocatalyst for use in biosensor development via an integrated enzyme-chemo catalytic cascade. This work sheds new lights on activating enzyme@MOF biocatalyst through a straightforward yet effective water impregnation process, paving the way for the design of next-generation biocatalysts with enhanced activity and stability.</p>