Recently, single-atom catalysts (SACs) have been used to construct biosensors for the determination of organophosphorus pesticides (OPs). However, most nanozymes including SACs are peroxidase-like enzymes and require highly toxic and unstable hydrogen peroxide (H2O2) as a co-reactant to generate reactive oxygen species. Inspired by the heme site of cytochrome c oxidases (Ccos), the construction of Fe-N5-coordinated SACs by introducing axial N ligands is expected to bind O2 to generate active metal-oxygen intermediates. Herein, a SAC with an Fe-N5 active center confined by hierarchically porous carbon nanoframes (Fe SAs/N5-pC-4) was prepared by a polymerization-pyrolysis-evaporation-etching strategy, and its underlying enzyme-like mechanism was uncovered through experiments and density functional theory calculations. The 100% metal atom utilization, increased accessible active sites, accelerated mass transfer, excellent hydrophilicity, and an electron-driven mechanism of axial N endow the SAC with enhanced oxidase-like activity. Notably, its catalytic rate constant (0.398 s-1) is 569 times greater than that of the commercial Pt/C catalyst. Similar to the catalytic mechanism of Ccos, O2 can be converted into reactive oxygen species, avoiding the use of co-reactant H2O2 effectively. In addition, based on the inhibitory effect of thiols on the active site of Fe SAs/N5-pC-4, a biosensor was constructed and applied to the colorimetric analysis of OPs. This provides a facile, cost-effective method for efficient OP screening at sites to help control their contamination.