Insulinomas are the primary etiology of endogenous hyperinsulinemic hypoglycemia, which often manifest with Whipple’s triad and neuroglycopenic symptoms. Given the diverse clinical manifestation and subtle onset of insulinomas generally in a small size, detecting a minority of these generally small tumors can be challenging. We reported a case of a 44-year-old female patient with recurrent hypoglycemia accompanied by hyperinsulinemia, and the conventional imaging revealed no abnormality. With the aid of endoscopic ultrasound-guided fine-needle aspiration biopsy (EUS-FNAB), the insulinoma was precisely diagnosed and localized, and successfully excised via operation. The patient’s hyperinsulinemia and hypoglycemic episodes were relieved significantly after surgery. The application of EUS-FNAB notably enhances the diagnostic accuracy for occult insulinomas, thereby informing appropriate surgical management. Herein, we advocate for invasive EUS examination in patients exhibiting strong clinical and laboratory indicators of insulinoma, even when conventional imaging results are negative.