Objective To construct an adult-derived thyroid organoid (ADTO) model and optimize its culture protocol in vitro. Methods Normal thyroid tissue from adult patients with papillary thyroid cancer (PTC) was digested and separated to obtain thyroid cells. The cells were embedded with Matrigel. Different conditioned culture media were designed and added. The forming efficiency and budding number of the first- and second-generation organoid were observed under microscope. Hematoxylin-eosin (H-E) staining was used to observe the histological morphology of the organoid model; immunohistochemical staining was used to detect the expression of thyroid specific markers, including NK2 homeobox protein 1 (NKX2.1), paired-box 8 (PAX8), and thyroglobulin (TG), to evaluate the consistency between the organoid model and normal thyroid tissue. Thyroxine (T4) and triiodothyronine (T3) levels in the supernatant of the ADTO models were detected by enzyme-linked immunosorbent assay (ELISA) to verify whether they have hormone secretion function or not. Results Eight ADTO models were successfully established from the surgical specimens of 7 PTC patients. In the ADTO culture system, after increasing the concentrations of Noggin and epidermal growth factor and combination of multiple small molecule inhibitors, the first- and second-generation organoid models had more buds, larger spheres, and higher efficiency of organoid formation. H-E staining showed that a large number of follicular structures surrounded by monolayer cubic epithelial cells could be seen in the ADTO model, which was similar to the structural characteristics of human thyroid tissue. Immunohistochemical staining showed positive NKX2.1, PAX8, and TG, which was consistent with the primary tissue. The results of ELISA showed T3 and T4 in the supernatant of the ADTO models, indicating that it had thyroid hormone secretion function. Conclusion The ADTO model established from human thyroid tissue is stable, which provides insights into regenerative medicine to treat hypothyroidism.