The invention relates to a novel fluorescence-marked vitamin B12 derivative, and concretely relates to a vitamin B12 derivative marked with fluorescein isothiocyanate (FITC), and the derivative is called a VB12-FITC for short. A preparation method of the VB12-FITC comprises the following steps: a diamino group is coupled with a low-reaction-activity hydroxyl group in a VB12 structure, and then the obtained aminated VB12 is reacted with an isothiocyanate radical of the FITC, so that the VB12-FITC derivative is synthesized. The advantages of the FITC-marked VB12 derivative are as follows: preparation conditions are gentle (the preparation can be performed at room temperature), raw materials are low in toxicity, the operation method is simple and convenient, the synthetic efficiency is high, the properties of reaction products are stable, and the FITC-marked VB12 derivative has good a fluorescence imaging effect. The FITC-marked VB12 derivative provided by the invention has a tumor-uptake targeting property and an important value in research of VB12 absorption and metabolic pathways, tumor imaging therapy and the like.