We demonstrate a new scheme for hydrogen peroxide (H2O2) detection through a distinctive fluorescence “on/off/on” mechanism. Specifically, we show that the fluorescence emission of carbon dots (C-dots) prepared from L-penicillamine (LPA) as the carbon precursor underwent initial quenching upon addition of hemoglobin (“off” state) but could be completely recovered upon addition of H2O2 (fluorescence “on”). The “on/off/on” phenomenon was observed only in case of C-dots prepared from penicillamine, underscoring a unique radical-scavenging mechanism by thiol residues upon the C-dots’ surface. This study illustrates a novel C-dot fluorescence tuning through heme oxidation and demonstrates a useful sensing modality for oxidative targets. © 2018 Elsevier B.V.