Objective: To investigate the diagnosis and surgical treatment of insulinoma. Methods: The clinical data of 72 patients with insulinoma admitted to our hospital spanning more than 31 years were retrospectively analyzed. Results: Of the 72 patients, 84.72% had a typical Whipple’s triad, and the ratio of plasma immunoreactive insulin to glucose (IRI/G) was greater than 0.3 in 80.56%. The positive rates of preoperative niveau diagnosis were 78.13% by transabdominal ultrasonography, 60.97% by CT, 100% by pancreatic perfusion imaging of multislice spiral CT, 37.50% by MRI, 72.73% by endoscopic ultrasound and 28.57% by DSA, respectively. The diagnostic positive detection rate of intraoperative ultrasonography (IOUS) plus palpation was 92.31%. The diameters of 88.75% of the tumors were less than 2 cm, and 37.5% of tumors were located in the head and neck of the pancreas, 27.5% in the body, and 35.0% in the tail, and 81.58% of the patients had indications for regional enucleation of insulinoma. All of the 72 cases were confirmed as insulinoma by final pathology, of whom 65 (90.28%) were functional insulinoma, 7 (9.72%) were nonfunctional insulinoma, 4 (5.56%) were multiple insulinoma, 2 (2.78%) had malignant tendency, and 1 (1.39%) was combined with multiple endocrine neoplasia type 1 (MEN-1). Conclusion: Whipple’s triad and IRI/G>0.3 can be the main basis for the defining diagnosis of insulinoma. A combination of multiple examinations such as ultrasonography, CT, pancreatic perfusion imaging of multislice spiral CT, MRI, endoscopic ultrasound and DSA may be recommended before operation, and IOUS plus palpation is a simple and effective way for intraoperative localization. Regional enucleation of insulinoma is the best surgical approach for most insulinoma.