Hydrogen peroxide (H2O2), a crucial biomarker of reactive oxygen species (ROS), is pivotal in diagnosing and treating cancer. Due to H2O2’s low concentration, fast diffusion, and natural degradation, traditional sensing single-readout methods have issues reliably monitoring and quantifying the cancer cells. Herein, to improve accuracy and an efficient way to boost the signals and increase the sensitivity, a dual-mode electrochemical (EC) and electrochemiluminescence (ECL) sensing system for monitoring H2O2 release from cancer cells in real-time has been developed using in-situ self-assembly of polycrystalline manganese oxide nanoflowers (cubic Mn2O3 and orthorhombic Mn3O4, MnxOy NFs) on the Ti3C2 MXene nanosheets. Peroxidase mimics of MnxOy/Ti3C2 MXenes serve not only as a co-reaction accelerator but also as a substrate for immobilizing high loading of luminol (LUM), which accelerates the generation of ROS and decreased the distance between the LUM and the generated OH. The hollow layered structure of Ti3C2 MXene as well as the synergistic effect of strong interaction between MnxOy NFs and Ti3C2 MXenes enhanced the heterostructures’ exceptional catalytic performance. The sensor demonstrated a unique 1.5 s current response for H2O2 detection, with an electrochemistry-wide linear response ranging from 0.05 to 650 μM and an ultrasensitive ECL limit of detection of 0.45 nM. The biosensor is remarkably effective in measuring the H2O2 released from breast cancer (MCF-7) under ascorbic acid simulation with a dynamic range of 100–6000 cell numbers, a low detection limit (20 cells), desirable anti-interference characteristics, and substantial capability to distinguish cancer cells from normal cells.