Abstract: Near-infrared fluorescence (NIRF) imaging is an emerging technology that aids in real-time intraoperative navigation. NIRF uses fluorescent dyes and probes to obtain real-time anatomical and functional information of blood and lymphatic vasculature and specific tissues during surgery. It can identify small cancerous growths and tumor margins that cannot be normally detected by traditional methods, thus providing the surgeons with a perioperative, anatomical guidance and identification modality to make correct intraoperative decisions. NIRF is promising owing to the following aspects:real-time, sensitive, safe, non-invasive, relatively cheap, and non-ionizing; it has the potential to bring a revolution in the existing surgical techniques. This article comprehensively reviews the basic principles of NIRF imaging technology, the clinical progress in real-time guided tumor resection, sentinel lymph node mapping for cancer staging, and protection of critical anatomical structures during surgery. Furthermore, this review discusses the advantages and limitations of NIRF technology and potential future prospects for its application.