The present study reports a sensor for the determination of glutathione using a carbon-paste electrode modified with reduced graphene oxide (RGO) and 10,10-dimethyl-7(3,4-dihydroxyphenyl)-10,11-dihydrochromeno[4,3-b]chromene-6,8(7H,9H)-dione (DDDC) as a mediator. Graphene nanosheets were obtained through the chemical reduction of graphene oxide using hydrazine. A pair of well-defined DDDC redox peaks was obtained at the modified electrode by direct electron transfer between DDDC and CPE. The novel sensor revealed suitable electrocatalytic activity toward the oxidation of glutathione (Glu). Voltammetric peak currents showed a linear response for Glu in the range of 0.08–100 μM with a detection limit (based on 3s_bl/m) of 0.02 μM. The results exhibited an efficient catalytic activity of the electrode for the electro-oxidation of Glu, which led to a reduction in its over-potential by more than 507 mV. In addition, voltammetric investigations of Glu and penicillamine (PA) showed two separate peaks at the modified electrode, thus the simultaneous determination of Glu and PA in the real samples was applied at the proposed electrode.