A novel mercury-doped silver nanoparticles film glassy carbon (Ag/MFGC) electrode was prepared in this study. Electrochemical behaviors of cysteine on the Ag/MFGC electrode were investigated by electrochemical impedance spectroscopy and cyclic voltammetry (CV). The results indicated that cysteine could be strongly adsorbed on the surface of the Ag/MFGC electrode to form a thin layer. The doped electrode could catalyze the electrode reaction process of cysteine, and the cysteine displayed a pair of well-defined and nearly reversible CV peaks at the electrode in an acetate buffer solution (pH 5.0). The Ag/MFGC electrode was used for determination of cysteine by differential pulse voltammetry. The linear range was between 4.0 × 10 −7 and 1.3 × 10 −5 mol/L, with a detection limit of 1.0 × 10 −7 mol/L and a signal-to-noise ratio of 3. The relative standard deviation was 2.4% for seven successive determinations of 1.0 × 10 −5 mol/L cysteine. The determinations of cysteine in synthetic samples and urinal samples were carried out and satisfactory results were obtained. Amperometric application of the Ag/MFGC electrode as biosensors is proposed.