To date, NIR fluorophores have been specifically designed for many different applications, including tumor detection, sentinel lymph nodes mapping, neurological diseases, cardiovascular diseases, bile duct identification, perfusion assessment, gland visualization, etc. For now, only Indocyanine Green (ICG), Methylene Blue (MB) and 5-Aminolevulinic acid have received FDA approval for clinical use. With the development of the devices, the major limitation in fluorescence image-guided surgery is the availability of clinically approved fluorescent dyes. Research made by different groups focus on the design of the chemical structure and optical properties of a fluorophore to meet the requirements for an imaging agent in surgery. This review focuses on the near-infrared (NIR) fluorophores used in different clinical applications of fluorescence image-guided surgeries in the last decade, lists their advantages and limitations for intraoperative use, outlines the key points to develop and optimize the imaging system, in order to issue a framework to make this technology more mature for future medicine.