Abstract: The present disclosure provides a fluorescently-traceable amino acid derivative and a preparation method and use thereof, and belongs to the technical field of biomedicine. In the present disclosure, an amino acid skeleton of the amino acid derivative is modified mainly by a fluorescently-traceable functional group. 4-chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl) has low polarity and strong fluorescence, and can be used to modify an N-terminal of the amino acid skeleton to conduct subcellular imaging. Moreover, the NBD-Cl has a relatively small volume, lacks reaction orthogonality, and causes little interference with biochemical reactions of the organism itself. The results of examples show that the fluorescently-traceable amino acid derivative has a desirable biological activity and can be fluorescently traced in vivo and in vitro.