Background and Aims In recent years, there has been a gradual increase in the adoption of day-surgery of laparoscopic cholecystectomy (DSLC). Given that patients usually undergo admission, surgery, and discharge within 24 h, DSLC demands higher standards of safety and efficiency. Moreover, excessive visceral fat in some overweight obese patients may affect the anatomical structure of the extrahepatic bile ducts during DSLC. Indocyanine green (ICG) fluorescence navigation may aid in identifying bile duct structures during DSLC, thus preventing bile duct injuries. Currently, there are few reports on the application of ICG fluorescence navigation in DSLC. Therefore, this study was performed to investigate the clinical application value of ICG fluorescence navigation in DSLC.Methods The clinical data of 55 patients who underwent DSLC in Zhejiang Provincial People's Hospital from July 2021 to October 2023 were retrospectively analyzed. Patients were divided into a fluorescence surgery group (22 cases) and a conventional surgery group (33 cases) based on whether ICG fluorescence navigation was used during operation. The general data, operative time, time for dissection of the Calot's triangle, estimated blood loss, and identification rate of extrahepatic bile ducts were compared between the two groups, and the impact of overweight obesity (BMI≥24 kg/m²) on the identification of extrahepatic bile ducts was also analyzed.Results There were no significant differences in general data, proportion of overweight obese patients, operative time, time for dissection of the Calot's triangle, and estimated blood loss between the fluorescence surgery group and the conventional surgery group (all P>0.05). Before dissection of the Calot's triangle, the identification rates of the cystic duct and common bile duct in the fluorescence surgery group were significantly higher than those in the conventional surgery group (59.1% vs. 21.2%, P=0.009; 50.0% vs. 18.2%, P=0.018). After dissection of the Calot's triangle, the identification rates of the common bile duct, hepatic duct, and cystic duct-common bile duct junction in the fluorescence surgery group were significantly higher than those in the conventional surgery group (100.0% vs. 69.7%, P=0.004; 100.0% vs. 33.0%, P<0.000 1; 86.4% vs. 27.3%, P<0.000 1). In the fluorescence surgery group, the identification rate of the common bile duct in overweight obese patients was significantly lower than that in non-overweight obese patients before dissection of the Calot's triangle (50.0% vs. 92.8%, P=0.039), while there was no significant difference in the identification rate of extrahepatic bile ducts between them after dissection of the Calot's triangle (P>0.05). Comparison between overweight obese patients in the fluorescence surgery group and the conventional surgery group showed that there was no significant difference in the identification rate of extrahepatic bile ducts between the two groups before dissection of the Calot's triangle (P>0.05),while the identification rates of the hepatic duct and cystic duct-common bile duct junction in the fluorescence surgery group were significantly higher than those in the conventional surgery group after dissection of the Calot's triangle (100.0% vs. 25.0%, P=0.001; 87.5% vs. 16.7%, P=0.005).Conclusion The application of ICG fluorescence navigation in DSLC is safe and feasible; it can improve the identification rate of extrahepatic bile ducts during DSLC, and even in overweight obese patients, greatly assist surgeons in clearly identifying extrahepatic bile ducts.