OBJECTIVE To investigate the differential diagnosis between Graves's disease and Hashimoto's hyperthyroidism to improve positive diagnostic rate in Hashimoto's hyperthyroidism, to facilitate proper management. METHODS Cytological examination was conducted by fine needle biopsy (FNB) on the thyroids of patients with hyperthyroidism diagnosed by clinical data, including 22 cases of Hashimoto's hyperthyroidism, 5 males and 17 females, aged 39 +/- 12, and 49 cases of Graves's disease, 20 males and 29 females, aged 41 +/- 11. Thyroid auto-antibodies-thyroid peroxidase antibody (TPOAb), thyroid microsomal antibody (TMA), thyroglobulin antibody (TGA), thyroid-stimulating hormone receptor antibody (TRAb), and free serum triiodothyronine (FT3), free thyroxin (FT4), and serum thyroid-stimulating hormone (S-TSH) were tested. The correlation between the pathology of thyroid and different autoantibodies and thyroid hormone was analyzed. RESULTS TRAb was 42.28 +/- 75.89 in Graves disease group, significantly higher than that in Hashimoto's hyperthyroidism group (5.77 +/- 12.69, P < 0.001). FT3 was 16.13 +/- 9.95, significantly higher than that in Hashimoto's hyperthyroidism group (9.54 +/- 6.76, P < 0.01). In Graves's disease group the positive rates for TRAb, TPOAb, TMA, and TGA were 83.67%, 75.51%, 57.14% and 48.98% respectively. In Hashimoto's hyperthyroidism group, the positive rate for TRAb was 13.64%, and the positive rates for the rest autoantibodies were all 86.36%. The double positive rate of TPOAb and TGA was 86.36% in Hashimoto's hyperthyroidism group, significantly higher than that in Graves's disease group (48.98%). There was a positive association between TRAb and FT3, and between diffuse and symmetric increased size of thyroid and FT3 TRAb. CONCLUSIONS Comprehensive analysis of TRAb, TPOAb, TGA, and FT3 is helpful to the differential diagnosis between Graves's disease and Hashimoto's hyperthyroidism.