This study focused on the development of chitosan and CNTs enhanced glassy carbon electrodes (CS/CNT/GCE) as a methadone electrochemical biosensor and its application to athlete doping testing in biological and food samples. SEM, FTIR, and XRD analysis results indicated the successful synthesis of CS/CNT-modified GCE. An electrochemical study of modified electrodes using the methods of cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and differential pulse voltammetry (DPV) revealed an improvement in the sensitivity, stability, and selectivity of the CS/CNT/GCE within methadone determination due to a synergistic effect between CS and CNT. The CS/CNT/GCE showed a robust and broad linear range of 40 to 1000 µM, a sensitivity of 0.03204 µA/µM, and a detection limit of 0.019 µM for methadone detection. In this investigation, genuine pear samples and blood serum samples from five athletes were utilized to examine the application of the created methadone sensor, and the results showed substantial recovery values (above 96.00%) and good precision (RSD value less than 4.76%). These results demonstrated that the CS/CNT/GCE sensor was dependable and precise for measuring methadone concentrations in biological and food samples.