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The application provides carbon nanodots with red anti-Stokes luminescence characteristics and a preparation method and application thereof, relates to the field of carbon nanomaterials, and the carbon nanodots are prepared by using o-phenylenediamine as raw material, mainly have a main emission peak in a red light-near infrared region, and have anti-Stokes emission characteristics in a protic solvent. The anti-Stokes luminescence of the carbon nanodots with red anti-Stokes luminescence characteristics is derived from phonon-assisted single-photon absorption, can be excited by a continuous laser light source, and the anti-Stokes luminescence intensity increases with the increase of temperature. The carbon nanodots with red anti-Stokes luminescence characteristics can be applied to anti-Stokes fluorescence imaging and optoelectronic devices as anti-Stokes imaging reagents.