- Detection of reactive oxygen species (ROS) is important in varied biological processes, disease diagnostics, and chemotherapeutic drug screening. We constructed a ROS sensor comprising an ascorbic-acid-based hydrogel encapsulating luminescent amphiphilic carbon- dots (C-dots). The sensing mechanism is based upon ROS-induced oxidation of the ascorbic acid units within the hydrogel scaffold; as a consequence, the hydrogel framework collapses resulting in aggregation of the C-dots and quenching of their luminescence. The C- dot–hydrogel platform exhibits high sensitivity and detected ROS generated chemically in solution and in actual cell environments. We demonstrate application of the C-dot–hydrogel for evaluating the efficacy of a chemotherapeutic substance, underscoring the potential of the system for drug screening applications.