Abstract Chylothorax is a serious complication after esophagectomy and an important reason for reoperation and prolonged hospital stay. A clear localization of thoracic ducts (TDs) and the potential leakage site is an ideal strategy to prevent postoperative chylothorax. In order to explore the feasibility and safety of near-infrared fluorescence imaging (NIR-FI) with indocyanine green (ICG) to identify TDs and potential chyle leakage during video-assisted thoracoscopic esophagectomy, a prospective clinical trial was conducted. We recruited 41 patients who underwent thoraco-laparoscopic minimally invasive esophagectomy for esophageal cancer in this prospective, single-arm clinical study between December 2020 and December 2021. The primary endpoint was the performance of NIR-FI in identifying TDs and chyle leakage. The ICG was injected subcutaneously into the right inguinal region 30 minutes before the thoracic operation, and it becomes fluorescent when excited by the light with a special wavelength in the NIR system. According to the intraoperative demands, surgeons can observe the position of the TDs under the real-time guidance of NIR-FI. In 38 of 41 patients (92.7%) using NIR-FI, TDs were clearly visible in high contrast. The mean signal-to-background ratio (SBR) of all fluorescent TDs was 3.05 ± 1.56. Fluorescence imaging of TDs could be detected 0.5 hours after ICG injection and last up to 3 hours with an acceptable SBR value. The optimal observation time window is from about 1 to 2 hours after ICG injection. Under the guidance of real-time NIR-FI, three patients were found to have chylous leakage and the selective TD ligation were performed intraoperatively. No patient had postoperative chylothorax. NIR-FI with ICG can provide highly sensitive and real-time assessment of TDs, as well as determine the source of chyle leakage, to prevent TD injury and direct selective TD ligation. It’s an effective navigation tool to minimize the incidence of chylothorax after minimally invasive esophagectomy.