Objective Hypothyroidism is a serious endocrine disease and an independent risk factor for systemic diseases. Studies of hypothyroidism can't be done without establishing animal models. C57BL/6 and KunMing (KM) mice are ideal animal vectors for the study of endocrine and other diseases. However, there are few studies describing total thyroidectomy in mice in detail. Methods In this study, total thyroidectomy was performed in C57BL/6 and KM mice by ligation (operation method I) and hemostasis (operation method II), and the detailed operation process was recorded. Elisa was used to detect and compare the serum TT3, TT4 and TSH levels, body weight and neck tissue HE staining of mice before and after surgery to verify the model. Results The serum TT3 and TT4 were decreased (P< 0.05) and TSH was increased (P< 0.001) in the two model groups. The survival rate within 28 days after operation in group I and Group II was 40% and 60%, respectively. In KM mice, it was 50% and 40%. The body weight of the two groups of mice was significantly higher than that of the sham group. HE staining and microscopic observation showed that the cervical tissue of the two species of mice was thyroid tissue, and the back membrane of the thyroid was intact after isolation. Conclusion Both surgical methods can cause hypothyroidism in both types of mice. However, it is necessary to be familiar with the anatomical relationship of mouse thyroid gland and surrounding tissue, improve the proficiency of surgical operation, prevent the occurrence of postoperative hypocalcemia and infection, and thus improve the survival rate of mice after modeling.