Rational modification of catalysts at electrode surface is essential and vital to regulate the electrochemically catalytic capability. In this work, we established an electrochemical interface functionalized by NiPd nanoparticles (NiPd NPs) mediated by metal–organic frameworks (MOFs) of Ni3HHTP2 ([email protected]3HHTP2) towards assaying H2O2 in living cells. In contrast to NiPd NPs, [email protected]3HHTP2 exhibited greatly enhanced electrocatalytic activity towards the reduction process of H2O2 with an onset potential of 0.10 V (vs. Ag/AgCl), as well as the current density increased by 23%. Moreover, the cathodic peak current showed a good linear relationship with H2O2 concentration in a wide range of 1.0 μM to 45.0 mM with a detection limit of 0.8 μM. The developed electrochemical method demonstrated good selectivity towards various potential interferences in cell system, and good reproducibility with a current deviation of 2.1% for 10 different functionalized electrodes. Eventually, the present electrochemical sensor with high selectivity and good stability was employed to monitor the levels of H2O2 released from living cells under oxidative stress.