The invention discloses a near-infrared window fluorescent imaging agent containing diazosulfide and a fluorene ring and a preparation method thereof. A modifiable group is introduced into the fluorene ring of the fluorescent compound, increased modifiable sites can be used for connection of different bioactive substances, water solubility and biological compatibility can be improved, and the application range in the biomedical field can be expanded. The fluorescent imaging agent has the advantages of high fluorescence intensity, no toxicity, good biocompatibility, and the like, and has excellent application prospects. The invention also discloses the application of the fluorescent imaging agent in the field of brain glioma, systemic angiography and sentinel lymph node dissection. In addition, the imaging agent has good modificability, and also can be used for in-vitro detection of various disease markers, in-vivo diagnosis and surgical navigation treatment of breast cancer, prostate cancer and colon cancer and other cancer, evaluation of curative effect after tumorectomy, and the like.