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Single-atom nanozymes (SAzymes) have drawn ever-increasing attention due to their maximum atom utilization efficiency and enhanced enzyme-like activity. Herein, a facile pyrolysis strategy is reported for the synthesis of the iron–nitrogen–carbon (Fe-N-C) SAzyme using ferrocene trapped within porous zeolitic imidazolate framework-8 (ZIF-8@Fc) as a precursor. The as-prepared Fe-N-C SAzyme exhibited exceptional oxidase-mimicking activity, catalytically oxidizing 3,3′,5,5′-tetramethylbenzidine (TMB) with high affinity (Km) and fast reaction rate (Vmax). Taking advantage of this property, we designed two colorimetric sensing assays based on different interaction modes between small molecules and Fe active sites. Firstly, utilizing the reduction activity of ascorbic acid (AA) toward oxidized TMB (TMBox), a colorimetric bioassay for AA detection was established, which exhibited a good linear range of detection from 0.1 to 2 μM and a detection limit as low as 0.1 μM. Additionally, based on the inhibition of nanozyme activity by the thiols of glutathione (GSH), a colorimetric biosensor for GSH detection was constructed, showing a linear response over a concentration range of 1–10 μM, with a detection limit of 1.3 μM. This work provides a promising strategy for rationally designing oxidase-like SAzymes and broadening their application in biosensing.