The concept of localization by imaginary clock for ampulla of gallbladder is definited to ensure security of laparoscopic cholecystectomy. According to the direction of the line of sight, three kinds of positioning method including vertical plane, horizontal plane and inclined plane is introduced. Ampulla of gallbladder could be classified into five types including 3 o’clock location, 6 o’clock location, 9 o’clock location, 12 o’clock location and axial view in the face of the dial plate. Ampulla of gallbladder in 3 o’clock location is more likely to be punctured, with a potential risk of injury to the common hepatic duct, right hepatic duct and right hepatic artery by excessive manipulation. Ampulla of gallbladder in 6 o’clock location might fall into Winslow hole and push the common bile duct to the ventral and left side. Dissection along the left and caudal edge of dilated ampulla, the common bile duct located the ventral side of ampulla might be misunderstood as the cystic duct to be cut off. Ampulla of gallbladder in 9 o’clock location might be adhere to duodenum seriously. Exenteration of the corresponding gallbladder wall is mandatory to avoid injuring duodenum. Ampulla of gallbladder in 12 o’clock location is frequently covering the ventral side of the common hepatic duct and the common bile duct. The common bile duct might be misunderstood for the cystic duct to be transected if dissecting along the left and caudal edge of ampulla. Localization by imaginary clock for ampulla of gallbladder could help to identify the anatomy correctly, avoid injuring extrahepatic bile duct and blood vessels, reduce bleeding, decrease iatrogenic cystic duct calculi and iatrogenic choledocholithiasis, overcome the significant difficulties of laparoscopic cholecystectomy safely.