Recent years have seen a growing interest towards real-time tracking and quantification of reactive oxygen species such as, H2O2 released in living cells, as they are potential biomarkers for pathogenesis. Herein, we described a rapid and sensitive enzymeless H2O2 assay using Prussian blue microcubes (PB MCs) decorated graphenated carbon nanotubes (g-CNTs) modified electrode for the tracking of in-vivo H2O2 production in mammalian cells. The hydrothermally prepared PB MCs were blended with g-CNTs to yield 3D hierarchical network of PB MCs/g-CNTs nanocomposite, suitable for efficient electrocatalysis. The g-CNTs/PB MCs film modified electrode was fabricated which displayed excellent electrocatalytic activity to H2O2 reduction at minimized overpotential. A straightforward amperometric sensor was constructed that displayed working range of 25 nM–1598μM, and detection limit of 13nM. The described non-enzymatic H2O2 assay is rapid, sensitive, selective, durable, reproducible and robust. The method was successful in sensing in-vivo H2O2 production from Raw 264.7 cells indicating its potential in biochemical and clinical applications.