The invention discloses a telomere probe and a preparation method and application thereof. According to the telomere probe, a single stranded DNA is coupled onto the surface of the CdSSe/ZnS quantum dots, the telomere probe can be combined with the cell telomere by in-situ hybridization technique, and the telomere hybridized with the telomere probe is imaged by a single molecular positioning technique to realize the detection of the telomere. The telomere probe realizes the specific marking of the telomere, has higher resolution due to the adoption of the single molecule positioning microscopeimaging, and overcomes the limitation by the optical diffraction limit of the fluorescence microscopic technique. The telomere probe and the preparation method and the application thereof can be usedfor further study of the telomere structure.