A DNA nanoprobe, activated by glutathione (GSH), was designed to enable spatially selective sensing and imaging of miRNA in living cells. The nanoprobe was constructed using nano-sized metal-organic frameworks (MOFs) and DNA hairpin probes tethered to the surface of the MOFs, with the loop portion of the hairpin structure containing a disulfide bond. Cleavage of the disulfide bond by GSH triggers a strand-displacement reaction with target miRNAs, facilitating in situ readout of the fluorescence signal. The synergy of endogenous GSH activation and MOF improves the spatial resolution of miRNA detection and imaging.