An indirect and sensitive electrochemical method for the determination of reduced glutathione (GSH) was developed by using poly(thionine) (PTH) as a mediator in the presence of Fenton-type reaction on the electrode surface in this work. Cyclic voltammetry and the adsorption of Cu2+ were employed in sequence to fabricate a Cu-PTH modified electrode, which was characterized by scanning electron microscopy, XPS and electrochemical measurements. Hydroxyl radicals that were derived from the Fenton-type reaction between Cu2+ and H2O2 could effectively oxidize PTH, leading to the great enhancement of the cathodic peak current of the dye polymer in the cyclic voltammetric scan. The electroreduction of PTH on the electrode surface was found to be inhibited in the presence of GSH. Under the optimized conditions, the cathodic peak current change was found to be proportional to the logarithm of the GSH concentration from 10nM to 1mM with a detection limit of 2.5nM estimated at a signal-to-noise ratio of 3. The proposed electrochemical sensor was successfully employed for the determination of GSH in real samples with satisfactory results.