Nitric oxide (NO) constitutes one of the most versatile therapeutics for biomedical applications. The efficient and on‐demand NO generation essentially dictates its concentration dependent therapeutic activity. Here, we report an electrochemical system employing a rationally designed single‐iron atom based biomimetic catalyst (i.e., Fe SAC), for high‐efficient and controlled NO generation. The Fe SAC shows superior catalytic ability in electrochemical reduction of nitrite, with maximal NO generation rate achieving 2.1 μM (min μg)−1. Theoretical studies suggest the significant decrease of Gibbs‐free energy of NO2− adsorption on single‐iron atom accounts for its high catalytic efficiency. Moreover, ample amount of NO can be controllably generated in a potential dependent manner. For antibacterial application, the generated NO overwhelmingly disrupts both gram‐negative and gram‐positive strains, highlighting a great promise to expedite NO research in both basic and applied sciences.