AbstractA zeolitic imidazolate framework (ZIF‐8) containing two enzymes that form a cascade biocatalyst has been integrated with 3D structures that are fabricated through a two‐photon polymerization using direct laser writing lithography. Glucose oxidase (GOx) and horseradish peroxidase (HRP) are encapsulated by the biomimetic self‐assembly process of ZIF‐8 to form the GOx/HRP/ZIF‐8 composite which grows in situ on the surface of microprinted carbon‐coated hexagonal substrates (cHS). The GOx/HRP/ZIF‐8/cHS film is applied to glucose detection through enzymatic oxidation of glucose to gluconic acid, forming H₂O₂, in the presence of GOx and the subsequent reduction of H₂O₂ to water in the presence of HRP and Amplex Red. This reaction is monitored using fluorimetry as the oxidation of Amplex Red with H₂O₂ catalyzed by HRP forms red‐emitting resorufin. The GOx/HRP/ZIF‐8/cHS film responds to glucose concentrations with a linear range of 10–200 µm, which correlates to the salivary glucose level in healthy humans. The GOx/HRP/ZIF‐8/cHS film shows more than eight times and 13 times enhanced activity than GOx/HRP/cHS (without ZIF‐8) and GOx/HRP/ZIF‐8 on a non‐3D patterned substrate, respectively. The GOx/HRP/ZIF‐8/cHS film retains more than 80% or 100% of its initial activity after being stored for over 2 months at ≤24 or 4 °C, respectively.