The electrocatalytic oxidation of glutathione based on rutin,as a mediator was studied at acetylene black (AB) modified carbon paste electrode (CPE). The results of cyclic voltammetric experiments showed that rutin could catalyze oxidation of GSH in PBS (pH 7.0) and the oxidation current was greatly increased by using AB-CPE compared to the nonmodified CPE. The kinetic parameters such as catalytic rate constant and diffusion coefficient were obtained using double potential step chronoamperometry. Under the optimum conditions, the electrocatalytic oxidation peak current was linear to the glutatbione concentration range from 0.5 mu M to 3.5 mu M and 2.5 mu M to 25 mu M with the detection limit of 0.080 mu M (S/N = 3) by differential normal pulse voltammetry (DNPV). The proposed method has been applied to the determination of glutathione in pharmaceutical sample with satisfactory results. (C) 2015 Elsevier B.V. All rights reserved.