Abstract A new fluorescent probe, nitroolefin functionalized diketopyrrolopyrrole (DPP-NO2) was designed and synthesized as a highly sensitive and selective probe for detecting glutathione (GSH) over cysteine (Cys), homocysteine (Hcy) and other amino acids in aqueous media. The probe is a non-fluorescent compound. On being mixed with GSH in buffered THF/PBS (v/v = 3:2) solution at pH 7.2, the probe exhibited the yellow-green emission at 548 nm and a 2000-fold enhancement in fluorescence intensity, along with a 26 nm hypsochromic shift from 515 nm to 489 nm in the absorption spectra. This probe was very sensitive and displayed a linear fluorescence off–on response to GSH with fluorometric detection limit of 6.14 × 10−8 M. What’s more, through the fluorescence turn-on signal response to GSH, the probe was successfully utilized for monitoring GSH in living cells.