This study aimed to investigate the feasibility and efficacy of near-infrared fluorescence-guided laparoscopic anatomical hepatectomy (LAH) using a novel indocyanine green (ICG)-human serum albumin complex (HSA) in patients with hepatocellular carcinoma. Clinical data of hepatocellular carcinoma patients who underwent ICG-HSA fluorescence-guided LAH at our center from January 2024 to April 2024 were prospectively collected and analyzed. Ultraviolet absorption spectroscopy was used to test the absorption and stability of ICG-HSA complex solutions under different conditions. After determining the optimal ratio, the complex was administered intravenously during surgery to perform negative staining via Glissonean pedicle isolation. LAH was performed along the fluorescence-demarcated boundaries. Thirty-one patients were included (24 men; mean age, 54.61 ± 13.54years). The median maximum tumor diameter was 2.80 (interquartile range [IQR], 2.00-4.00) cm. S8 segmentectomy (22.6%) and right posterior segmentectomy (19.4%) were the most common resections performed. Successful fluorescence negative staining was achieved in all patients using ICG and HSA at a 1:6 molar ratio at room temperature. Mean operation time was 297.58 ± 85.53min, Median intraoperative blood loss was 100.0mL (IQR, 50.0-200.0). The median surgical margin distance was 0.90cm (IQR, 0.40-1.50). The postoperative complication rate was 45.2% (35.5% Clavien-Dindo grade I and 9.7% grade II). The median length of hospital stay was 5.0days (IQR, 4.0-5.0). ICG-HSA-assisted LAH is safe and feasible. Compared with free ICG, the novel ICG-HSA complex exhibits better optical properties and in vivo stability, which can improve the accuracy of intraoperative liver segment localization and optimize the anatomical dissection plane. It has the potential to become an ideal fluorescent imaging agent for anatomical hepatectomy.