Biliary injury is a severe complication that can be associated with liver surgery. Intrahepatic biliary anatomy can be evaluated using magnetic resonance cholangiopancreatography and X-ray cholangiography; however, an intraoperative real-time bile duct visualization method has not yet been reported. This study aimed to demonstrate the availability of real-time fluorescent cholangiography (FC) by intrabiliary indocyanine green administration with near-infrared laparoscopy in major hepatectomy. The optimal concentration of indocyanine green (ICG) solution was examined ex vivo. The fluorescence intensity of the ICG solution and its mixture with bile was measured. Using a clinical trial model, ICG solution was injected into the cystic duct, followed by near-infrared laparoscopy performed during hepatectomy. The optimal concentration of ICG solution for FC was between 0.01 and 0.05 mg/mL. Three different laparoscopic systems were used in three hepatectomy cases. In all cases, the fluorescence of the intrahepatic bile ducts in the Glissonian sheath was clearly visualized using the near-infrared laparoscopic system. A small piece of tissue prevented the bile glow; thus, exposure of the Glissonian sheath was necessary for clear FC. This procedure also detected bile leakage from the cut surface of the liver. Intrabiliary ICG administration and near-infrared laparoscopy enabled real-time intrahepatic FC during major hepatectomy.