Objective: To study the significance of combined detection of serum anti-thyroglobulin antibody (TGAb), thyroid peroxidase antibody (TPOAb), and thyroid stimulating hormone (TSH) in the differential diagnosis of benign and malignant thyroid nodules (TNS). Methods: A total of 98 patients with TNS in our hospital from June 2023 to June 2024 were selected. All patients underwent serum TgAb, TPOAb and TSH tests. The diagnostic results and diagnostic efficacy of individual and combined tests of serum TgAb, TPOAb and TSH for benign and malignant nodules were analyzed. And compare the differences in serum TgAb, TPOAb and TSH levels between benign and malignant patients. Results: In this experiment, 98 patients with TNS were selected. Pathological examination showed that 41 cases were malignant and 57 cases were benign. TgAb examination showed that 31 cases were malignant and 51 cases were benign. The TPOAb examination showed that 30 cases were malignant and 50 cases were benign. The TSH examination showed that 26 cases were malignant and 51 cases were benign. The combined test showed that 38 cases were malignant and 55 cases were benign. The combined detection sensitivity was 92.67% (38/41) and the accuracy was 94.90% (93/98), which were compared with TgAb examination at 75.61% (31/41) and 83.67% (82/98), TPOAb examination at 73.17% (30/41) and 81.63% (80/98), and TSH examination at 63.41% (2) It was higher than 6/41 and 78.57% (77/98), and the missed diagnosis rate was 7.32% (3/41), which was lower than 24.39% (10/41) in TgAb examination, 26.83% (11/41) in TPOAb examination, and 36.59% (15/41) in TSH examination (P < 0.05). Compared with benign patients, the levels of serum TgAb, TPOAb and TSH in malignant patients were higher (P < 0.05). Conclusion: The combined detection of serum TgAb, TPOAb and TSH has relatively high sensitivity and accuracy in the differential diagnosis of benign and malignant TNS patients. It can also reduce the rate of missed diagnosis. Moreover, the levels of serum TgAb, TPOAb and TSH in patients with malignant TNS are significantly abnormal, which can provide a reference basis for formulating treatment plans.