In nature, individual cells contain multiple isolated compartments in which cascade enzymatic reactions occur to form essential biological products with high efficiency. Here, we report a cell-inspired design of functional hydrogel particles with multiple compartments, in which different enzymes are spatially immobilized in distinct domains that enable engineered, one-pot, tandem reactions. The dense packing of different compartments in the hydrogel particle enables effective transportation of reactants to ensure that the products are generated with high efficiency. To demonstrate the advantages of micro-environmental modifications, we employ the copolymerization of acrylic acid, which leads to the formation of heterogeneous multi-compartmental hydrogel particles with different pH microenvironments. Upon the positional assembly of glucose oxidase and magnetic nanoparticles, these hydrogel particles are able to process a glucose-triggered, incompatible, multistep tandem reaction in one pot. Furthermore, based on the high cytotoxicity of hydroxyl radicals, a glucose-powered therapeutic strategy to kill cancer cells was approached.Cells contain isolated compartments where cascade enzymatic biochemical reactions occur to form essential biological products with high efficiency. Here the authors produce functional hydrogel particles with multiple compartments via microfluidics that contain spatially immobilized natural enzymes in distinct domains for one-pot, tandem reactions.