Chemical modification with foreign atoms is a leading strategy to intrinsically modify the properties of host materials. Among them, potassium (K) modification plays a critical role in adjusting the electronic properties of carbon materials. Graphene, a true 2D carbon material, has shown fascinating applications in electrochemical sensing and biosensing. In this work, a facile and mild strategy to K-modifying in graphene at room-temperature is reported for the first time. X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), transmission electron microscopy (TEM), Raman spectra, and cyclic voltammetry are used to characterize this K-modified graphene. The K-modified graphene is capable of acting as an electron transfer medium and more efficiently promotes charge transfer than unmodified graphene. A highly sensitive and stable amperometric sensor based on its excellent electrocatalytic activity toward the oxidation of NO2− is proposed. The sensor shows a linear range from 0.5 μM to 7.8 mM with a detection limit of 0.2 μM at a signal-to-noise ratio of 3. The modified electrode has excellent analytical performance and can be successfully applied in the determination of NO2− released from liver cancer and leukemia cells and shows good application potential in biological systems.