Surgery is the only cure for many solid tumors, but positive resection margins, damage to vital nerves, vessels and organs during surgery, and the range and extent of lymph node dissection are significant concerns which hinder the development of surgery. The emergence of fluorescence-guided surgery (FGS) means a farewell to the era when surgeons relied only on visual and tactile feedback, and it gives surgeons another eye to distinguish tumors from normal tissues for precise resection and helps to find a balance between complete tumor lesions removal and maximal organ function conservation. However, the existing synthetic fluorescence contrast agent has flaws in safety, specificity and biocompatibility to various extents. Extracellular vesicles (EVs) are a group of heterogeneous types of cell-derived membranous structures present in all biological fluids. EVs, especially engineered targeting EVs, play an increasingly important role in drug delivery because of their good biocompatibility, validated safety and targeting ability. Nevertheless, few studies have employed EVs loaded with fluorophores to construct fluorescence contrast agents and used them in FGS. Here, we systematically reviewed the current state of knowledge regarding FGS, fundamental characteristics of EVs, and the development of engineered targeting EVs, and put forward a novel strategy and procedures to produce EVs-based fluorescence contrast agent used in fluorescence-guided surgery.Copyright © 2022. Published by Elsevier B.V.