6-[18F]fluoro-L-DOPA is a radiotracer widely used in the diagnosis of a range of diseases, including neuro-oncology, endocrinology, and Parkinson’s disease. To meet the rapidly growing clinical need for this radioactive compound, this study reports an optimized radiosynthesis for this molecule, which proved to be highly reliable and compatible with different types of automated radiosynthesizers. Moreover, with 6-[18F]fluoro-L-DOPA, the PET/CT imaging of a total of 23 patients has been conducted, further demonstrating this radiotracer as a clinically valuable reagent to diagnose congenital hyperinsulinism (CHI) of infants in a non-invasive manner and, more importantly, localize the exact lesion on pancreas.