Elevated levels of Troponin I, a cardiac biomarker, is indicative of an Acute Myocardial Infarction. However, current immunosensing methods to detect the presence of Troponin I such as Enzyme-Linked Immunosorbent Assay (ELISAs) has poor detection accuracy and unreliable quantitative results due to its single model readout. On the other hand, impedance spectroscopy detection using aptamer-modified electrodes is a more reliable and robust detection method, with higher sensitivity and better selectivity. In this paper, we propose a molybdenum disulphide/cellulose acetate (MoS2/CA) nanofiber composite on screen printed electrodes for detecting troponin I by Electrical Impedance Spectroscopy. The MoS2/CA nanofiber was electrospin at various amount of MoS2 nanosheets (0.025 g, 0.05 g, 0.1 g and 0.2 g). Morphological, structural and optical characterization was conducted to discover the functional group as well as crystallinity formed in the MoS2/CA composite nanofiber. The developed electrochemical nano-biosensor can detect up to 10 fM of Troponin I with 90 stability after 6 weeks, ~ 5 folds improvement compared to other proteins (selectivity), 0.89 µA mM�1 cm� 2 sensitivity of sensor and RSD value of 3.8 (repeatability). © 2021, The Author(s), under exclusive licence to Springer Nature B.V.