The present disclosure discloses a fluorescent starch nanocrystal and a preparation method and application thereof. Mainly based on the character that starch nanocrystal has an active hydroxyl group on the surface, the present disclosure introduces fluorescein isothiocyanate into the surface of the starch nanocrystal in the form of stable covalent bonds through a two-step chemical modification method: silane coupling and electrophilic addition of fluorescein, so as to prepare the starch nanocrystal with fluorescent properties. The preparation process of the present disclosure is simple and easy to operate, uses cheap and readily available raw materials, involves low cost, and allows the non-fluorescent starch nanocrystal to produce significant fluorescent properties without affecting the properties of the starch nanocrystal itself, thereby expanding the application of starch. Moreover, the fluorescent starch nanocrystal thus obtained has significant fluorescent properties, good biocompatibility and biodegradability, has application prospects in the fields of biosensor preparation, bioimaging marking and fluorescent analysis and detection, and can be further prepared into a novel nano-fluorescent probe with organic nano-particles as the matrix.