Diabetic keratopathy (DK) is a common chronic metabolic disorder that causes ocular surface complications. Among various therapeutic approaches, local delivery of nerve growth factor (NGF) remains the most effective treatment of DK. However, achieving a sustained therapeutic effect with NGF and the frequent drug delivery burden remain challenging during clinical practice. Here, we developed a novel adeno-associated virus (AAV)-based NGF delivery system that achieved 1-year-long-lasting effects by a single injection. We refined the corneal stromal injection technique, resulting in reduced corneal edema and improved AAV distribution homogeneity. AAV serotype AAV.rh10 exhibited high tropism and specificity to corneal nerves. A dose of 2 × 109 vector genomes was determined to achieve efficient Ngf gene expression without inducing corneal immune responses. Moreover, NGF protein was highly expressed in trigeminal ganglion through a retrograde transport mechanism, indicating the capacity for repairing corneal nerve damage at both the root and corneal nerve endings. In a mouse DK model, a single injection of AAV-Ngf into the corneal stroma led to marked corneal nerve regeneration for over 5 months. Together, we provide a novel therapeutic paradigm for long-term effective treatment of DK, and this therapeutic approach is superior to current DK therapies.