In this work, a novel aptamer-based CA-MoS2 hybrid biosensor was developed for diagnosis of Acute Myocardial Infraction (AMI). Specifically, a novel petal-like MoS2 nanoflower was used as the active material on the Screen-Printed Electrode (SPE) for AMI biomarker determination. The aptamer-based CA-MoS2�24 hybrid was able to attain a low limit of detection (LOD) and sensitivity to 10 fM at a linear range from 10 fM to 1 nM with good reproducibility based on an electrochemical impedance spectroscopy and a ~4 folds higher selectivity of troponin I compared to the signal from other biomolecules in human serum, retaining a 91 stability after 6 weeks. This novel CA-MoS2 aptamer-based biosensor with a 2.3 RSD value has the potential to revolutionize medical diagnosis by conducting multiple biomolecule detection on a single sensing platform. © 2021 Taiwan Institute of Chemical Engineers