In this work, chemiluminescent (CL) property of the carbon dots in the presence of peroxynitrous acid was studied. Peroxynitrous acid is formed by online mixing of nitrite and acidified hydrogen peroxide. The CL intensity was increased linearly with nitrite concentration in the range from 1.0 × 10(-7) M to 1.0 × 10(-5) M, and the detection limit was 5.3 × 10(-8) M (signal-to-noise ratio of S/N = 3). This method has been successfully applied to the determination of nitrites in pond water, river water, and pure milk, with recoveries in the range of 98%-108%. The CL mechanism of the peroxynitrous acid-carbon dots system was investigated using the CL, ultraviolet-visible light (UV-vis), and electron paramagnetic resonance (EPR) spectra. The electron-transfer annihilation of hole-injected and electron-injected carbon dots could mainly account for the CL emission, which sheds new light on the optical properties of the carbon dots.