Catching cancer at an early stage is necessary to make it easier to treat and to save people's lives rather than just extending them. Reactive oxygen species (ROS) have sparked a huge interest owing to their vital role in various biological processes, especially in tumorigenesis, thus leading to the potential of ROS as prognostic biomarkers for cancer. Herein, a non-enzymatic biosensor for the dynamic monitoring of intracellular hydrogen peroxide (H 2 O 2 ), the most important ROS, via an effective electrode composed of poly (diallyldimethylammonium chloride) (PDDA)-capped reduced graphene oxide (RGO) nanosheets with high loading trimetallic AuPtAg nanoalloy, is proposed. The designed biosensor was able to measure H 2 O 2 released from different cancerous cells promptly and precisely owing to the impressive conductivity of RGO and PDDA and the excellent synergistic effect of the ternary alloy in boosting the electrocatalytic activity. Built upon the peroxidase-like activity of the nanoalloy, the developed sensor exhibited distinguished electrochemical performance, resulting in a low detection limit of 1.2 nM and a wide linear range from 0.05 µM to 5.5 mM. Our approach offers a significant contribution toward the further elucidation of the role of ROS in carcinogenesis and the effective screening of cancer at an early stage.It has been proven that reactive oxygen species (ROS), including superoxide (O 2− ), hydrogen peroxide (H 2 O 2 ), hydroxyl radicals, and peroxynitrite, are messenger molecules, which can turn various biological processes on and off [6]. Balanced ROS metabolism increases antioxidant ability, thereby establishing a barrier against tumorigenesis [7,8]. However, DNA damage caused by ROS results in genomic instability and altered gene expression, while ROS-mediated signaling pathways also drive cell proliferation and apoptosis suppression, thus promoting tumor formation [9,10]. Among the ROS, H 2 O 2 has sparked huge interest in anticancer therapies as not only is it the main substance in the cellular response to oxidative stress, but it is also able to pass through cell membranes over a relatively long lifetime. In addition, cancer cells have metabolic and signaling aberrations, and thus exhibit an enhanced H 2 O 2 level that differs from that in normal cells [11,12]. Therefore, the measurement of intracellular H 2 O 2 could be of great value in clarifying its role in oncogenesis and exploring advanced therapeutic strategies for cancer and other ROS-related diseases.Although various analytical techniques for measuring H 2 O 2 , such as spectrophotometry and chemiluminescence, have been developed, they still suffer drawbacks such as low detection limits and complex processing when faced with the challenge of achieving detection limits in the nanomolar range in living cells [13,14]. In this regard, electrochemical techniques with high sensitivity and simplicity to improve the timely detection of H 2 O 2 in vivo have gained increasing attention. Enzymes like horseradish peroxidase have been widely us...