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Hydrogen peroxide (H 2 O 2 ) has been reported to mediate a variety of physiological and pathological processes in living systems. In this work, a biosensor for determination of H 2 O 2 was prepared by using an HRP/Ti 3 C 2 /Nafion film-modified glassy carbon electrode (GCE). Ti 3 C 2 nanosheets with remarkable conductivity and high specific surface area were chosen as carriers for HRP. Moreover, this biosensor modified with HRP has a specific catalytic effect on H 2 O 2 . The difference in peak current could reflect the quantitative change of H 2 O 2 . The linear range of the biosensor is 5-8000 μM, and the detection limit is 1 μM ( S / N = 3). This biosensor was used to detect H 2 O 2 in clinical serum samples of normal controls and patients with acute myocardial infarction (AMI) before and after percutaneous coronary intervention (PCI). The results showed that the difference between normal controls and patients is significant ( P < 0.05), as well as the difference for patients before and after PCI ( P < 0.01), but no significant difference existed between postoperative patients and normal controls. This biosensor has the advantages of simple preparation, high sensitivity, and quick detection, showing potential application in clinical diagnosis.