We report the design and fabrication of a new type of nanohybrid microelectrode based on a hierarchical nanostructured Au/MnO2/graphene-modified carbon fiber (CF) via in situ electrochemical synthesis, which leads to better structural integration of different building blocks into the CF microelectrode. Our finding demonstrates that wrapping CF with graphene nanosheets has dramatically increased the surface area and electrical conductivity of the CF microelectrode. The subsequent template-free electrodeposition of MnO2 on graphene-wrapped CF gives rise to a porous nanonest architecture built up from twisted and intersectant MnO2 nanowires, which serves as an ideal substrate for the direct growth of Au nanoparticles. Owing to the structural merit and synergy effect between different components, the hierarchical nanostructured noble metal/metal oxide/graphene-coated CF demonstrates dramatically enhanced electrocatalytic activity. When used for nonenzymatic H2O2 sensing, the resultant modified microelectrode exhibits acceptable sensitivity, reproducibility, stability, and selectivity, which enable it to be used for real-time tracking H2O2 secretion in human cervical cancer cells. Graphical abstract A schematic illustration of preparation of hierarchical Au/MnO2/ERGO/CF nanohybrid electrode for real-time molecular detection of cancer cells.