The study of cellular responses linked to oxidative stress mechanisms is crucial in comprehending diverse physiological and pathological life processes, including mitochondrial dysfunction. Nonetheless, despite the interference of O2, the monitoring of ROS released from cells poses a challenging task. In this study, carbon-based copper single-atom catalysts (Cu SACs) were synthesized that exhibits excellent electrocatalytic performance for H2O2 reduction with an initial potential at 0.23 V and effectively avoids interference from O2. Based on this catalyst, a flexible and stretchable oxygen-tolerant sensor was constructed and applied to monitor the calcium ion-induced ROS burst in human umbilical vein endothelial cells (HUVECs) in a simulated physiological condition. This study effectively eradicates interference that may arise from the reduction of O2 and presents a dependable platform for real-time in situ monitoring of physiologically active molecules by utilizing H2O2 detection.