Enzymatic cascade reactions with high activity and specificity in living cells always benefit from multicompartmentalized organelles that provide separately confined spaces for enzymes, avoiding their mutual interference to ensure the high-efficiency operation of necessary vital movements. Inspired by this, we designed a 3D spherical microreactor (Au@H-APF@Pt) with biomimetic cascade catalysis for glucose detection. First, ultrasmall gold nanoparticles were immobilized in situ on the internal cavities of hollow 3-aminophenol formaldehyde resin (H-APF) nanospheres, along with glucose oxidase activity. Then, platinum nanoparticles (PtNPs) with peroxide-like activity were reduced surrounding the outer layer of the H-APF nanospheres. Similar to the cell structure, different metal sites in this bifunctional microreactor operated independently, bringing higher catalytic activity and selectivity and thus being synergistically capable of a cascade reaction to catalyze the substrate for glucose detection. This cell-mimicking microreactor (Au@H-APF@Pt) was successfully applied in glucose colorimetric detection, showing a 1.9-fold activity enhancement compared to direct mixing (Au/Pt). The observed low catalytic activity was attributed to the extended time for transferring hydrogen peroxide (H2O2) from Au NPs to the solution and then to PtNPs. Integrating a smartphone APP, a real-time, visual, and Au@H-APF@Pt-based hydrogel sensor for glucose detection was also proposed. Satisfactory results highlight that this cell-mimicking microreactor offers a very successful strategy to improve the efficiency of cascade catalysis systems in biosensing.