Objective To evaluate the security and clinical value of the combination of three-dimensional computed tomography-bronchography and angiography (3D-CTBA) and indocyanine green (ICG) staining in video-assisted thoracic surgery (VATS) segmentectomy. Methods The clinical data of 125 patients who received VATS segmentectomy from January 2020 to January 2021 in our hospital were retrospectively analyzed. There were 40 (32.0%) males and 85 (68.0%) females with an average age of 54.8±11.1 years. Results The procedure was almost identical to the preoperative simulation. All intersegment planes were displayed successfully by ICG reverse staining method. There was no allergic patient. A total of 130 pathological specimens were obtained from the 125 patients. The mean operation time was 126.8±41.9 min, the time of first appearance of fluorescence was 22.7±4.9 s, the mean mark time was 65.6±20.3 s, the median blood loss was 20.0 (10.0-400.0) mL, the postoperative hospital stay was 5.6 (4.0-28.0) d, and the postoperative retention of chest tube time was 3.2 (2.0-25.0) d. Pathological results showed that microinvasive adenocarcinoma was the most common type (38.5%, 50/130), followed by invasive adenocarcinoma (36.9%, 48/130); there were 3 metastatic tumors (3/130, 2.3%).Conclusion The combination of 3D-CTBA and ICG reverse staining is proved to be a safe, necessary and feasible method. It solves the difficult work encountered in the procedure of segmentectomy, and it is worth popularizing and applying in clinic.