Graphene film has been considered a promising material for the construction of self-supported electrodes due to its favorable flexibility and high conductivity. However, the film fabricated from pristine graphene or conventional graphene sheet reduced graphene oxide processes limited electrocatalytic performance. Decorating active metal species or incorporating heteroatoms intothegraphene framework have been proved to be effective methods to enhance the electrocatalytic efficiency of graphene film-based self-supported electrodes. Herein, we present a freestanding electrode composed of uniform Pd nanoparticles decorating N,S co-doped porous graphene film (Pd/NSPGF) and explore its practical application in differentiating various human colon cell types by in situ tracking the amount of H2O2 secreted from live cells. Our findings reveal that, on the one hand, the NSPGF has abundant surface and inner pores, which promote active site exposure, and mass diffusion during electrochemical reactions; on the other hand, the substitutional doping ofthe graphene framework with heteroatoms (e.g., N or S) can tailor its electronic and chemical properties, and facilitate the uniform loading of high-density Pd nanoparticles. Moreover, the intrinsic activity of Pd/NSPGF is regulated by the interaction of Pd nanoparticles withtheNSPGF support. Taking the advantages of morphology and composition, the self-supported Pd/NSPGF electrode displays remarkable electrochemical performance with a wide linear range up to 2.0mM, low detection limit of 0.1μM (S/N = 3), high sensitivity of 665 µA cm-2mM-1, and good selectivity. When applied in real-time trackingofthe H2O2 released from normal human colon epithelial cells and human colorectal cancer cells, the Pd/NSPGF-based electrochemical sensing system can distinguish the cell types by testing the number of extracellular H2O2 molecules released per cell, which holds considerable potential for early detection and monitoring of disease-related clinical specimens.