H₂O₂ plays a role as a signaling molecule in normal bodily processes and serves as an important marker for oxidative stress in the development and progression of atherosclerosis and other cardiovascular diseases. However, the quantification of H₂O₂ in in vitro cell lines and in vivo cardiac tissue has been challenging, hindered by limitations in sensitivity, selectivity, and the need for rapid, non-invasive detection methods. This study successfully developed and validated the rGO-Co₃O₄-Pt/SPEs platform as a highly sensitive, selective, and robust biosensor for H₂O₂ detection, demonstrating its efficacy in both in vitro and in vivo settings. The biosensor effectively detects elevated ROS levels in DOX-treated cells and mice, demonstrating high selectivity and sensitivity in complex biological environments, minimizing false positives and enhancing cell viability and morphological alterations. Strong agreement with traditional fluorescence-based methods highlights the biosensor's accuracy and usefulness, successfully distinguishing between normal and stressed cells and tissues. The rGO-Co₃O₄-Pt/SPEs biosensor offers a robust foundation for advancing real-time ROS monitoring for advancing cardiovascular research, oxidative stress diagnostics, and the development of therapeutic interventions for ROS-related diseases.