We report a novel reaction-based thiol selective turn-on near-infrared (NIR) fluorescence and colourimetric dinitrobenzenesulfonyl-cyanine (DNBSCy) probe. In the presence of thiols such as glutathione (GSH), new absorption bands (476 and 581 nm) were observed, with the colour of the solution (10 mM PBS, pH = 7.4) changing from light green to blue. Interestingly, relatively non-fluorescent DNBSCy exhibited enhanced fluorescence emission around 700 nm in the NIR region. GSH reacted efficiently with the electron withdrawing sulfonyl ester moiety of DNBSCy, releasing the quinone embedded heptamethine cyanine (Cy-quinone) with extended π-electron conjugation responsible for the turn-on NIR fluorescence. Cy-quinone also displayed a conjugated π-electron push–pull character under physiological conditions. The DNBSCy probe was effectively employed to monitor the thiols in fetal bovine serum (FBS). The probe was capable of monitoring the oxidized glutathione (GSSG)/GSH redox process in the presence of glutathione reductase and NADPH with NIR fluorescence and colourimetric optical response. Thus, DNBSCy has the potential to measure the activity of glutathione reductase as a measure of oxidative stress.