Herein, we report the synthesis and characterisation of a new fluorescent probe 4-(7-nitro-benzo[1,2,5]oxadiazol-4-yl)-benzaldehyde (NBOB) installed with quenching groups for highly selective and sensitive sensing of biothiols. The probe itself is non-fluorescent due to the presence of quenching groups and photoinduced electron transfer (PET) process. Thus, sensitivity of the probe towards thiols was significantly improved by quenching effects. NBOB has been shown to exhibit selective reactivity towards cysteine (Cys) and homocysteine (Hcy) over glutathione (GSH) under stoichiometric conditions. The response mechanism was proved by 1H NMR, LCMS and theoretical calculation. The probe NBOB has been shown to react with Cys present in Vero cells by fluorescence microscopy. © 2016 Informa UK Limited, trading as Taylor & Francis Group.