A label-free biosensor is presented using carbon nanofiber (CNF) nanoelectrode arrays for the detection of cardiac troponin-I in the early diagnosis of myocardial infarction. Immobilization of anti-cTnI Ab on CNFs and the detection of human-cTnI were examined using electrochemical impedance spectroscopy and cyclic voltammetry techniques. Each step of the modification process was monitored, and the results show changes in electrical capacitance or resistance to charge transfer due to the specificity of corresponding adsorption of Ab-Ag interaction. The immunosensor demonstrates a good selectivity and high sensitivity against human-cTnI analytes and is capable of detecting cTnI at concentrations as low as ∼0.2 ng/mL, which is 25 times lower than that possible by conventional methods. Analysis of the electrode at various stages using atomic force microscopy and X-ray reflectivity provides information on the surface roughness and orientation of the antibody.