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Glutathione (GSH) serves many cellular functions and plays crucial roles in human pathologies. Simple and sensitive sensors capable of detecting GSH would be useful tools to understand the mechanism of diseases. In this work, a rapid fluorescence “switch-on” assay was developed to detect trace amount of GSH based on carbon dots–MnO 2 nanocomposites, which was fabricated through in situ synthesis of MnO 2 nanosheets in carbon dots colloid solution. Due to the formation of carbon dots–MnO 2 nanocomposites, fluorescence of carbon dots could be quenched efficiently by MnO 2 nanosheeets through fluorescence resonance energy transfer (FRET). However, the presence of GSH would reduce MnO 2 nanosheets to Mn 2+ ions and subsequently release carbon dots, which resulted in sufficient recovery of fluorescent signal. This proposed assay demonstrated highly selectivity toward GSH with a detection limit of 300 nM. Moreover, this method has also shown sensitive responses to GSH in human serum samples, which indicated its great potential to be used in disease diagnosis. As no requirement of any further functionalization of these as-prepared nanomaterials, this sensing system shows remarkable advantages including very fast and simple, cost-effective as well as environmental-friendly, which suggest that this new strategy could serve as an efficient tool for analyzing GSH level in biosamples. [ABSTRACT FROM AUTHOR]