We report a novel design, based on a combination of lanthanide-doped upconversion nanoparticles and manganese dioxide nanosheets, for rapid, selective detection of glutathione in aqueous solutions and living cells. In this approach, manganese dioxide (MnO2) nanosheets formed on the surface of nanoparticles serve as an efficient quencher for upconverted luminescence. The luminescence can be turned on by introducing glutathione that reduces MnO2 into Mn2+. The ability to monitor the glutathione concentration intracellularly may enable rational design of a convenient platform for targeted drug and gene delivery.