Determination of intracellular bioactive species will afford beneficial information related to cell metabolism, signal transduction, cell function, and disease treatment. In this study, the electrochemically reduced graphene oxide modified carbon fiber microdisk electrode (ER-GOME) was used as a detector of CZE-electrochemical detection and developed to detect glutathione (GSH). The electrocatalytic activity of the modified microelectrode was characterized by cyclic voltammetry. Under optimized experimental conditions, the concentration linear range of GSH was from 1 to 60 mu M. When the S/N ratio was 3, the concentration detection limit was 1 mu M. Compared with the unmodified carbon fiber microdisk electrode, the sensitivity was enhanced more than five times. With the use of this method, the average contents of GSH in single HepG2 cells were found to be 7.13 +/- 1.11 fmol (n = 10). Compared with gold/mercury amalgam microelectrode, which was usually used in determining GSH, the electrochemically reduced graphene oxide modified carbon fiber microdisk electrode was friendly to environment for free mercury. Furthermore, there were several merits of the novel electrochemical detector coupled with CE, such as comparative repeatability, easy fabrication, and high sensitivity, hold great potential for the single-cell assay.