Oxidative stress is a hallmark of a range of diseases including cancer, HIV-AIDS and cardiovascular disorders. Blood plasma cysteine (Cys) is an established biomarker for oxidative stress, rendering the development of sensitive and technically straightforward approaches for its estimation extremely desirable. Herein, we achieve this goal by developing a series of Michael addition–elimination–cyclization based turn-on fluorescence (MADELCY TOF) probes that selectively detect Cys over other amino acids and biothiols such as homocysteine (Hcy) and glutathione (GSH). Cysteine detection by these compounds involves cascaded fluorophore release via the Michael addition of the cysteinyl thiol followed by the elimination of a leaving group and lactamization via the cysteinyl amine. Conveniently, the Cys-reactivity of these probes can be tuned via both inductive and mesomeric effects imparted by their β′ position-substituents. One of our probes that contains the p-nitrophenyl group at its β′ position demonstrated rapid kinetics (t_1/2 = 2.9 min) with excellent sensitivity (detection limit = 8.2 nM) towards Cys. This probe also enabled the sensitive (detection limit = 9.5 pM) estimation of the blood plasma levels of the cancer and liver cirrhosis biomarker, the aminoacylase-1 (ACY-1) enzyme. Furthermore, another probe of our library enabled live cell imaging of cellular Cys allowing us to develop an imaging-based assay to monitor hydrogen peroxide-induced oxidative stress in mammalian cells.