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The unique chemical and physical properties of metal−organic frameworks (MOFs) endow them with promising applications in the electrochemical sensing field. Herein, a novel label-free electrochemical sensing strategy for cardiac troponin I (cTnI) has been constructed on the basis of immunoaffinityinduced depression of the electrochemical signal of Cu-MOF-74. The electrochemical assay shows that the immunoaffinity reaction between cTnI and anti-cTnI on the electrode surface can effectively inhibit the electrochemical signal of electrode-confined Cu-MOF-74, suggesting that the Cu-MOF-74-based interface may find application in the label-free analysis of cTnI. Thus, Cu-MOF-74 acts as a bifunctional material in this strategy, namely, the immobilization matrix of probe molecules and the signal source of the sensing analysis. In addition, the reason for the electrochemical signal depression of Cu-MOF-74 induced by immunoaffinity reaction is attributed to the increase in the insulation layer and the blocking of the electrolyte diffusion from the bulk solution to the electrode surface. In this strategy, the analytical range of cTnI is from 0.01 pg mL −1 to 1.0 ng mL −1 and the detection limit is 4.5 fg mL −1 . The Cu-MOF-74-based sensing interface is also reliable for cTnI analysis in real serum samples, showing that the biosensor has promising application in point-of-care testing (POCT) of myocardial injury-related diseases.