Segmentectomy is a targeted surgical approach tailored for patients with compromised health and early-stage lung cancer. The key to successful segmentectomy lies in precisely identifying the tumor and intersegmental planes to ensure adequate resection margins. In this study, we aimed to enhance this process by simultaneously visualizing the tumor and intersegmental planes through the intravenous injection of indocyanine green (ICG) at different time points and doses. Lung tumors were detected by intravenous injection of ICG at a dose of 2 mg/kg 12 h before surgery in a rabbit model. Following the dissection of the pulmonary artery, vein, and bronchi of the target segment, 0.6 mg/kg of ICG was injected intravenously to detect the intersegmental plan. Fluorescent images of the lung tumors and segments were acquired, and the fluorescent signal was quantified using the signal-to-background ratio (SBR). Finally, a pilot study of this method was conducted in three patients with lung cancer. In a preclinical study, the SBR of the tumor (4.4 ± 0.1) and nontargeted segments (10.5 ± 0.8) were significantly higher than that of the targeted segment (1.6 ± 0.2) (targeted segment vs. nontarget segment, p < 0.0001; target segment vs. tumor, p < 0.01). Consistent with preclinical results, lung tumors and the intersegmental plane were successfully detected in patients with lung cancer. Consequently, adequate resection margins were identified during the surgery, and segmentectomy was successfully performed in patients with lung cancer. This study is the first to use intravenous ICG injections at different time points and doses to simultaneously detect lung cancer and intersegmental planes, thereby achieving segmentectomy for lung cancer.