This study aimed to investigate the efficacy of different doses of Indocyanine Green (ICG) for fluorescence imaging during laparoscopic cholecystectomy, specifically between 0.5 to 3 h, to determine the optimal dose that would enhance clinical practice. A total of 80 patients with benign gallbladder diseases were enrolled fluorescence-guided laparoscopic cholecystectomy. Patients were randomly assigned to four groups based on the dose of ICG administered (n = 20 each): 0.25 mg, 0.50 mg, 1.00 mg, and 2.50 mg. Key clinical data were collected during the perioperative period, focusing on the delineation of the liver, identification of the cystic duct-common bile duct junction, and fluorescence intensity contrast analysis. The effectiveness of extrahepatic bile duct visualization was evaluated subjectively by three experienced hepatobiliary surgeons via surgical videos and images, categorized as “excellent,” “good,” or “poor.” Successful fluorescence imaging was achieved in all patients without the requiring conversion to open surgery. Significant differences were observed among the four groups in several metrics: intraoperative blood loss, bile duct identification time, fluorescence intensity contrast values, and the number of cases rated “excellent” by the surgeons (P < 0.05). No significant differences were observed for the remaining statistical indicators (P > 0.05). The findings based on the data suggest that a dose of 0.25 mg ICG within the 0.5–3 h window is optimal, yielding the highest fluorescence contrast between the extrahepatic bile duct and liver while garnering the best subjective evaluations. This study provides robust evidence to support the clinical application of ICG in surgical imaging, emphasizing its utility in clinical practice.