Background: We evaluated the use of thyroid stimulating immunoglobulin (TSI) assay to optimize the detection strategy for Graves' disease. Methods: Five hundred and forty-four well characterized serum samples from the Clinical Laboratory of Shanghai Tongren Hospital were collected from August 2019 to April 2020. The serum samples were obtained from 52 untreated GD patients, 155 treated GD patients, 83 patients with Hashimoto's thyroiditis, 70 patients with thyroid nodules, 83 patients with thyroid cancer, and 101 healthy subjects. All samples were evaluated by both TSI assay and TSH receptor autoantibodies (TRAb) assay. Moreover, 23 patients without a distinct thyroid disease diagnosis at the first visit were monitored for 6 months to make a final diagnosis. Results: The clinical sensitivity of the TSI and TRAb assays was 98.10% and 94.20% respectively, while the clinical specificity was 92.30% and 96.70% respectively. ROC plot analysis based on sera of UT-GD (newly diagnosed GD patients) revealed an area under the curve (AUC) of 0.974 for the TSI assay. The best cutoff value was 0.58 IU/l (98.0% of sensitivity, 92.8% of specificity). The AUC for the TRAb assay was 0.961. Furthermore, combining TSI and TRAb results, the area under the curve was 0.981. In a pilot study of 23 patients with an uncertain initial diagnosis, the follow-up results showed the clinical diagnosis of 22 out of 23 cases were resolved in agreement with the results obtained by the TSI assay, and one case matched the result obtained by TRAb assay. Conclusion: The TSI assay presents very promising analytical characteristics and could be adopted in clinical practice to improve GD diagnosis. The TSI assay might be better than TRAb assay in initial differential diagnosis of GD from other thyroid diseases.