Hydrogen peroxide (H 2 O 2 ) is an eminent biomarker in pathogenesis; a selective, highly sensitive real-time detection of H 2 O 2 released from live cells has drawn a significant research interest in bioanalytical chemistry. Binary transition-metal oxides (BTMOs) displayed a recognizable benefit in enhancing the sensitivity of H 2 O 2 detection; although the reported BTMObased H 2 O 2 sensor's detection limit is still insufficient, it is not appropriate for in situ profiling of trace amounts of cellular H 2 O 2 . In this paper, we describe an efficient, reliable electrochemical biosensor based on Mn 2 CuO 4 (MCO) microspheres to assay cellular H 2 O 2 . The Mn 2 CuO 4 microspheres were prepared through a superficial solvothermal method. It is obvious from impedance studies, introduction of manganese into copper oxide lattice significantly improved the ionic conductivity, which is beneficial for the electrochemical sensing process. Thanks to the distinct microsphere structure and excellent synergy, MCO-modified electrode exhibited excellent nonenzymatic electrochemical behavior toward H 2 O 2 sensing. The MCO-modified electrode delivered a broad working range (36 nM to 9.3 mM) and an appreciable detection limit (13 nM), with high selectivity toward H 2 O 2 . To prove its practicality, the developed sensor was applied in the detection of cellular H 2 O 2 released by RAW 264.7 cells in presence of CHAPS. These results label the possible appliance of the sensor in clinical analysis and pathophysiology. Thus, BTMOs are evolving as a promising candidate in designing catalytic matrices for biosensor applications. KEYWORDS: reactive oxygen species (ROS), H 2 O 2 , manganese copper oxide, electrocatalysis, electrochemical sensor