Research on the electro-oxidation of glutathione (GSH) may give insight into its real oxidation mechanism on biomembranes. In the present work, the unusual oxidation process of GSH on an Au electrode was probed by cyclic voltammetry (CV) technique. Voltammetric studies showed that –SH in GSH was the unique target of electro-oxidation by excluding the other potential oxidation sites (amido group, carboxyl group and side-chains of glutamic acid and glycine) and the feeble influence of S–Au interaction. As a result of spatial baffle, GSH was directly oxidized to GSOxH (x = 2, 3) without forming the intermediate of glutathione disulfide (GSSG). The unusual oxidation process differs from the twostep oxidation processes of cysteine –SH on the Au electrode and the oxidation of –SH in dissolved GSH, but is similar to the biological oxidation of GSH in vivo on biomembranes, where the steric hindrance still exists.