Acute myocardial infarction (AMI) is a major cause of death globally. Studies show that cardiac troponin I (cTnI) is an ideal serum biomarker for AMI diagnosis and its sensitive detection is of great value for the effective treatment of the disease. Here, a new electrochemical biosensor for sensitive and specific detection of cTnI is proposed based on the use of a multifunctional peptide-oligonucleotide conjugate. The conjugate is designed to consist of a peptide domain that can bind to cTnI with high specificity and a DNA domain that can mediate the templated growth of quantum dots (QDs) to serve as the source of signals. The presence of target cTnI leads to the recruitment of QDs onto magnetic beads, thereby producing a remarkable electrochemical response on screen-printed electrode. Experiential results show that the biosensor allows cTnI detection with a desirable sensitivity and specificity even in serum samples. The detection limit is calculated to be 0.42 pg/mL. More inspiringly, the biosensor is feasible for monitoring the drug-induced change of cTnI expression level on cardiomyocyte-like cells derived from mesenchymal stem cells (MSCs), opening a new way for investigating MSCs differentiation. Overall, the biosensor may provide a powerful tool for cTnI detection, which has great potential for practical use in cell biology research and clinical diagnosis.