Single-atom catalysts received increasing attention due to their maximum atom utilization efficiency. However, metal-free single atoms have not been used to construct electrochemical sensing interfaces. In this work, we demonstrated the use of Se single atoms (SA) as electrocatalyst for sensitive electrochemical nonenzymatic detection of HO. Se SA was synthesized and anchored on nitrogen-doped carbon (Se SA/NC) via a high-temperature reduction strategy. The structural properties of Se SA/NC were characterized by transmission electron microscopy (TEM), high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM), energy-dispersive spectroscopy (EDS), X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD), and electrochemical techniques. The results showed that Se atoms were uniformly distributed on the surface of the NC. The obtained SA catalyst exhibited excellent electrocatalytic activity toward HO reduction, and can be used to detect HO in a wide linear range from 0.04 mM to 11.1 mM with a low detection limit of 0.018 mM and high sensitivity of 403.9 µA mM cm. Moreover, the sensor can be used for the quantification of HO concentration in real disinfectant samples. This work is of great significance for expanding the application of nonmetallic single-atom catalysts in the field of electrochemical sensing. Se single atoms (Se SA) as novel electrocatalyst were synthesized and anchored on nitrogen-doped carbon (NC) for sensitive electrochemical nonenzymatic detection of HO.