Herein, a sensitive, selective, rapid and cost-effective fluorescence sensing platform has been designed for the detection of glutathione (GSH). In this platform, the manganese dioxide (MnO2) nanosheets served as both oxidant, which could effectively oxidize o-phenylenediamine (OPDA) to produce fluorescence for highly sensitive detection, and recognition element. When GSH was introduced, the MnO2 nanosheets were reduced to Mn2+, which inhibited the formation of fluorescent oxidation products of OPDA. Under optimal conditions, the MnO2-OPDA sensing platform showed very sensitive response to GSH in the range of 10 nM–40 μM. The detection limit of GSH reached as low as 10 nM. The high selectivity of inhibition of fluorescent product formation by GSH allows this platform to be used for detecting GSH in practical biological samples (such as cell extracts).