Using the spherical COFTAPB-DMTP as a carrier to sequentially load AuNPs, Abs, and HRP, a COF-supported signal probe was formed for the sandwiched EC immunosensing of cardiac troponin I (cTnI), a reliable biomarker of acute myocardial infarction, with a low LOD of 1.7 pg mL−1. This evidence concerns the gene TNNI3 and acute myocardial infarction.