Tripeptide glutathione (GSH) is an abundant and ubiquitous metabolite in living organisms that plays critical roles in various cellular bioprocesses. In this work, we report the development of a new nanoprobe (MnO₂-PEI-FITC) for GSH detection and imaging through exploiting the response mechanism of specific GSH-triggered reduction of manganese dioxide (MnO₂) nanosheet. The MnO₂-PEI-FITC nanoprobe was developed by coating negatively charged MnO₂ nanosheet with positively charged polyethylenimine (PEI) polymer, followed by coupling with fluorescein isothiocyanate (FITC) through a thiourea linkage. The MnO₂-PEI-FITC nanoprobe showed weak fluorescence due to the quenching of FITC emission by MnO₂’s absorption, while FITC’s emission at 518 nm was observed in the presence of GSH. The MnO₂-PEI-FITC nanoprobe featured rapid response to GSH (< 12 min), high sensitivity (detection limit, 164 nM) and selectivity. Application of this nanoprobe for GSH imaging in in fresh yeast cells and onion inner-layer epidermal tissues were then successfully demonstrated. This work thus provides a new nanoprobe for GSH detection and imaging in biological samples.