Nanozymes are increasingly used as components in assays and diagnostics. Here, we describe a rapid and highly sensitive colorimetric assay for the detection and quantification of glutathione (GSH) employing MnO2 nanosheets as an artificial oxidase. In the assay pale yellow 3,3',5,5'-tetramethylbenzidine (TMB) is oxidized to a blue product (oxTMB) under catalyzing of MnO2 nanosheets with a significant change in absorption at 650 nm. GSH selectively inhibits this reaction with a detection limit of 300 nM. The high specificity of inhibition by GSH allows this system to be used to determine the GSH concentrations in human serum samples. The MnO2 nanosheet-based assay is simple, rapid, sensitive and selective for the quantification of GSH and surpasses detection methods based on other MnO2 nanomaterials.