Highly evolved multi-enzyme cascade catalytic reactions in organisms facilitate rapid transfer of substrates and efficient conversion of intermediates in the catalytic unit, thus rationalizing their efficient biocatalysis. In this study, pore-ordered mesoporous single-atom (Fe) nitrogen-doped carbon nanoreactors (Mp-Fe-CN) were designed, in which a reasonable pore size was designed as a natural enzyme trap coupled to a simulated enzyme center. A polarity-mediated strategy was developed to obtain atomically dispersed nanoporous substrates, with the finding that polarity-guided engineering of the nitrogen-ligand environment and vacancy cluster defects clearly affects nanoporous activity, accompanied by appreciable mesoporous pore size elevation. The active center and distal N atom coordination of Fe-N₄ affect the catalytic process of the nanozyme exposed by density functional theory (DFT), determining the contribution of hybridized orbitals to electron transfer and the decisive step. A cascade nanoreactor-based domain-limited sarcosine oxidase developed for non-invasive monitoring of sarcosine levels in urine for evaluation of potential prostate carcinogenesis as a proof of concept. Based on the design of surface mesoporous channels of nanocatalytic units, a bridge was built for the interaction between nanozymes and natural enzymes to achieve cascade nanocatalysis of natural enzymatic products.