Reactive oxygen species (ROS)‐based therapeutic strategies play an important role in cancer treatment. However, in situ, real‐time and quantitative analysis of intracellular ROS in cancer treatment for drug screening is still a challenge. Herein we report one selective hydrogen peroxide (H2O2) electrochemical nanosensor, which is prepared by electrodeposition of Prussian blue (PB) and polyethylenedioxythiophene (PEDOT) onto carbon fiber nanoelectrode. With the nanosensor, we find that the level of intracellular H2O2 increases with NADH treatment and that increase is dose‐dependent to the concentration of NADH. High‐dose of NADH (above 10 mM) can induce cell death and intratumoral injection of NADH is validated for inhibiting tumor growth in mice. This study highlights the potential of electrochemical nanosensor for tracking and understanding the role of H2O2 in screening new anticancer drug. A H2O2‐selective nanoelectrode is reported to monitor the dynamics of H2O2 produced by NADH treatment, indicating the potential tumor suppressor role of NADH. This work not only provides a potential therapeutic strategy for cancer therapy, but also offers a powerful technique to screen the cancer treatment.