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It is crucial to develop sensitive and accurate sensing strategies to detect H₂O₂ and glucose in biological systems. Herein, biocompatible iron-coordinated L-lysine–based hydrogen peroxide (H₂O₂)-mimetic enzymes (Lys-Fe-NPs) were prepared by precipitation polymerization in aqueous solution. The coordinated Fe²⁺ ion acted as centers of peroxidase-like enzymes of Lys-Fe-NPs, and the catalytic activity was evaluated via the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) by H₂O₂. Therefore, a sensitive colorimetric detection sensor for H₂O₂ was constructed with a linear range of 1 to 200 μM and a detection limit of 0.51 μM. The same method could also be applied to highly sensitive and selective detection of glucose, with a linear range of 0.5 to 150 μM and a detection limit of 0.32 μM. In addition, an agarose-based hydrogel biosensor colorimetric was successfully implemented for visual assessment and quantitative detection of glucose. The design provided a novel platform for constructing stable and nonprotein enzyme mimics with lysine and showed great potential applications in biorelevant assays.