© 2021 by the authors. Licensee MDPI, Basel, Switzerland.In this study, novel carbon nanotube-supported Mo (Mo/CNT) catalysts were prepared with the sodium borohydride reduction method for the detection of L-cysteine (L-Cys, L-C). Mo/CNT catalysts were characterized with scanning electron microscopy with elemental dispersion X-ray (EDX-SEM), X-ray diffraction (XRD), UV-vis diffuse reflectance spectrometry (UV-vis), temperature-programmed reduction (TPR), temperature programmed oxidation (TPO), and temperature-programmed desorption (TPD) techniques. The results of these advanced surface characterization techniques revealed that the catalysts were prepared successfully. Electrochemical measurements were employed to construct a voltammetric L-C sensor based on Mo/CNT catalyst by voltammetric techniques such as cyclic voltammetry (CV) and differential pulse voltammetry (DPV). Further measurements were carried out with electrochemical impedance spectroscopy (EIS). Mo/CNT/GCE exhibited excellent performance for L-C detection with a linear response in the range of 0–150 µM, with a current sensitivity of 200 mA/µM cm2 (0.0142 µA/µM), the lowest detection limit of 0.25 µM, and signal-to-noise ratio (S/N = 3). Interference studies showed that the Mo/CNT/GCE electrode was not affected by D-glucose, uric acid, L-tyrosine, and L-trytophane, commonly interfering organic structures. Natural sample analysis was also accomplished with acetyl L-C. Mo/CNT catalyst is a promising material as a sensor for L-C detection.