A novel nano-composite of graphene quantum dot-β-cyclodextrin was fabricated on the surface of glassy carbon electrode (GCE) using one step and green electrodeposition method. The redox of l-Cys behavior prepared nano-compositemodified of GCE was then characterized by cyclic voltammetry, chronoamperometry, and differential pulse voltammetry. Voltammograms of the modified electrode are recorded in physiological pH (phosphate buffer solution, pH 7.4), the kinetics of charge transfer and mass transport processes across the nano-composite/solution interface were studied. The modified electrode showed an efficient electrocatalytic activity toward the oxidation of l-Cysteine (l-Cys) through a surface-mediated electron transfer. The catalytic rate constant and the l-Cys diffusion coefficient were reported. A sensitive and time-saving method (differential pulse voltammetry) was developed for the analysis of l-Cys. The proposed voltammetric method was also applied to the determination of l-Cys using graphene quantum dot-β-cyclodextrin-GCE.