A simple amino-functionalization method for carbon nanotubes and its application in an electrochemical immunosensor for detection of the human cardiac troponin T are described. Amino-functionalized carbon nanotubes allow oriented antibodies immobilization via their Fc regions, improving the performance of an immunosensor. Herein multiwalled carbon nanotubes were amino-functionalized by using the ethylenediamine reagent and assays were designed by fractional factorial study associated with Doehlert matrix. Structural modifications in the carbon nanotubes were confirmed by Fourier transform infrared spectroscopy. After amino-functionalization the carbon nanotubes were attached to screen-printed carbon electrode and a sandwich-type immunoassay was performed for measuring the cardiac troponin T. The electrochemical measurements were obtained through hydrogen peroxide reaction with peroxidase conjugated to the secondary antibody. Under optimal conditions, troponin T immunosensor was evaluated in serum samples, which showed a broad linear range (0.02 to 0.32 ng mL−1) and a low limit of detection, 0.016 ng mL−1. This amino platform can be properly used as clinical tool for cardiac troponin T detection in the acute myocardial infarction diagnosis.