To evaluate the efficacy and safety of indocyanine green (ICG)-guided near-infrared fluorescence (NIRF) imaging during surgery to diagnose the cause of neonatal cholestasis (NC). Data on NC patients who underwent both NIRF with ICG and conventional laparoscopic bile duct exploration (the gold standard) at our institute from January 2022 to December 2022 were retrospectively analyzed. The patients’ baseline characteristics and liver function outcomes were collected and analyzed, and the diagnostic consistency was compared between the 2 methods. In total, 16 NC patients were included in the study, comprising 8 (50%) male and 8 (50%) female patients, ranging in age from 42 to 93 days, with a median age of 54.4 ± 21 days. During surgery, all the patients underwent NIRF with ICG, followed by conventional laparoscopic bile duct exploration. Finally, 15 of the patients were diagnosed with biliary atresia (BA) (1 with type-I BA, and 14 with type-II BA). The other patient was diagnosed with cholestasis. The diagnostic results from fluorescence imaging with ICG were consistent with those from conventional laparoscopic bile duct exploration. ICG-guided NIRF is associated with an easy operation, less trauma, and good safety. Also, its diagnostic accuracy is similar to conventional laparoscopic bile duct exploration.