Background and Aims Duodenum-preserving pancreatic head resection (DPPHR) is a commonly used option for benign or low-grade malignant space-occupying lesions of the head of the pancreas. Despite advances in laparoscopic techniques, performing this procedure under traditional laparoscopic approaches is still considerably challenging. In recent years, the emergence of 3D laparoscopic techniques overcomes the shortcomings of traditional laparoscopic methods and makes laparoscopic surgery more precise and safer. Here, the authors summarize the experience in 4 cases undergoing 3D laparoscopic DPPHR guided by fluorescence imaging, so as to provide a reference for the application and promotion of this technique.Methods The clinical data of 4 patients who underwent 3D laparoscopic laparoscopic DPPHR under fluorescence imaging navigation from August 2019 to May 2021 in the People's Hospital of Xiangxi Tujia and Miao Autonomous Prefecture were analyzed retrospectively.Results The 3D laparoscopic DPPHR under fluorescence imaging navigation was successfully completed in all 4 patients. The operative time was 370-510 min, and the blood loss was 80-300 mL, with no transfusion requirements of red blood cells and plasma. After the operation, one patient developed delayed gastric emptying, which was resolved by inserting a nasojejunal nutrition tube, 2 patients developed grade A biochemical leakage, and there were no complications such as duodenal perforation, postoperative bile leakage, postoperative bleeding, and secondary operation. All 4 patients were followed up for 3-24 months and recovered well.Conclusion Performing 3D laparoscopic DPPHR guided by fluorescence imaging is safe and feasible with the appropriate selection of patients in an experienced pancreatic surgery center.