Background: Tear deficiency due to dysfunction of the lacrimal gland is one of the major causes of dry eye disease (DED). Artificial tears as conventional medicine often alleviate symptoms, however, they insufficient to prevent the progression of severe DED. Lacrimal gland stem cell-based therapies have been extensively reported to regenerate injured lacrimal tissue, but the number of stem cells in the lacrimal gland is pretty low, and 2D long-term cultivation results in the reduction of the differentiation capacity of stem cells. Therefore, 3D lacrimal gland organoids could be an alternative for a DED therapeutic method, which is capable of prolonged growth while maintaining the characteristics of the lacrimal gland tissue.Method: Digested cells from human lacrimal gland tissue were mixed with Matrigel and cultured in five different media modified from human prostate/salivary organoid culture media. After organoid formation, the growth, expression of specific markers and histological characters were analyzed to prove the lacrimal gland organoids. And then, the secretory function of lacrimal gland organoids was confirmed by calcium influx or proteomics after pilocarpine treatment.Result: Human lacrimal gland organoids were formed and cultured under a modified salivary organoid culture medium, and their histological features and gene expression pattern were similar to normal lacrimal gland tissue. After pilocarpine treatment, their secretory function was confirmed and the secretomes were analyzed by proteomics. More than 70% of the identified proteins were proven to exosome through GO analysis. Additionally, to explore the curability of developed organoids, mouse-derived lacrimal gland organoids were fabricated and transplanted into mouse lacrimal tissue with DED, and we confirmed that organoids were well engrafted in the lacrimal tissue.Conclusion: These results demonstrate that our established lacrimal gland organoids resemble characteristics of normal lacrimal gland tissue and could be used as cell therapy for patients with dry eye syndrome.