<p>Hydrogen peroxide (H₂O₂) is a key reactive oxygen species involved in physiological regulation and pathological processes, and its accurate quantification is essential for bioanalysis and disease-related studies. Herein, we report the synthesis of a series of Cu-Fe oxide nanozymes via a simple antisolvent crystallization strategy and systematically investigate their peroxidase-like catalytic activity toward colorimetric H₂O₂ detection. By tuning the Cu/Fe molar ratio, the catalytic activity of the nanozymes can be effectively regulated, with Cu₂FeO_x exhibiting the highest peroxidase-like activity toward 3,3′,5,5′-tetramethylbenzidine (TMB) oxidation in the presence of H₂O₂ under acidic conditions and enabling sensitive colorimetric detection of H₂O₂ over a linear range of 10–200 µM with a detection limit of 0.572 µM. Furthermore, the proposed sensing platform demonstrates satisfactory selectivity and reproducibility, and is successfully applied to the determination of H₂O₂ released into cell culture supernatants after chemical stimulation. This work highlights the potential of composition‑regulated Cu-Fe oxide nanozymes as robust artificial enzymes for colorimetric H₂O₂ sensing and provides insights into their application in bioanalytical systems.</p>