Cobalt nanoparticle modified indium tin oxide (CoNP/ITO) electrodes fabricated by ion implantation were applied for the detection of reduced glutathione (GSH). The CoNP/ITO electrode was characterized by scanning electron microscopy (SEM), energy dispersive X-ray (EDX) detector and X-ray photoelectron spectroscopy (XPS). The assay performance with regard to GSH were evaluated by cyclic voltammetry (CV) and chronoamperometry (I-t). The proposed sensor exhibited a much higher electrocatalytic activity toward the oxidation of GSH than the bare ITO electrode, with a detection limit of 5 nM. The CoNP/ITO electrode showed enhanced electrocatalytic properties, high sensitivity, good long-term stability and reproducibility as well as a rapid response to detect GSH. In addition, the CoNP/ITO electrode was also used to analyse the GSH concentration in eye drop samples, and the results were in good agreement with the labelled values.