Hydrogen peroxide (H 2O 2) is known to play a multifaceted role in cell physiology mechanisms involving oxidative stress and intracellular signal transduction. Therefore, the development of analytical tools providing information on the dynamics of H 2O 2generation remains of utmost importance to achieve further insight in the complex physiological processes of living cells and their response to environmental stress 1. In the present work we developed a novel optic biosensor that provides continuous real-time quantification of the dynamics of the hydrogen peroxide release from cells under oxidative stress conditions. The biosensor is based on the ultra-sensitive dark field optical detection of cytochrome c (cyt c) that exhibits a narrow absorption peaks in its reduced state (Fe(II)) at λ= 550 nm. In the presence of H 2O 2the ferrous heme group Fe(II) is oxidised into Fe(III) providing the spectroscopic information exploited in this approach. Extremely low limit-of-detection for H 2O 2down to the subnanomolar range is achieved by combining scattering substrates (eg. polystyrene beads) able to shelter cyt c and an inverted microscope in dark field configuration. The developed biosensor was able to perform real-time detection of H 2O 2extracellular release from human promyelocytic leukemia cells (HL-60) exposed to lipopolysaccaride (LPS) that elicits strong immune-response. This biosensing tool is currently being implemented to the real-time detection of superoxide anion (O 2 . -) and offers the possibility to extend to further oxidative stress biomarkers such as glutathione. More generally, multianalyte and dynamic informations might bring new insights to understand complex cellular metabolisms involved in oxidative-stress-related diseases and cytotoxic responses. © 2012 SPIE.