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Hydrogen peroxide (H2O2), a key reactive oxygen species (ROS), plays a crucial role in cellular signaling; however, at elevated concentrations, it contributes to oxidative stress and is implicated in various pathologies. Herein, we report the development of a novel electrochemical biosensor based on cerium–hemin metal–organic frameworks (Ce–hemin–MOFs ) integrated with graphene oxide (GO) for the sensitive and selective detection of H2O2. The Ce–hemin–MOFs were synthesized via a coordination-driven assembly of cerium ions and hemin, yielding petal-like crystalline microstructures with intrinsic peroxidase-mimicking activity. Incorporation of GO significantly enhanced the electrical conductivity of the composite. The sensor demonstrated a broad linear detection range (0.01–10 mM), a low detection limit of 1.2 μM, and strong selectivity against common biological interferents. Furthermore, the developed sensor enabled real-time detection of H2O2 released from human prostate cancer (22Rv1) cells, demonstrating its practical potential for monitoring oxidative processes associated with cellular pathophysiology. This highlights the broader applicability of MOFs-based sensing platforms in biomedical research and disease diagnostics.