The aim of this study was to determine the trimester-specific reference range of thyroid function in Nanjing.A total of 805 pregnant women in the 1st, 2nd, and 3rd trimesters were recruited in the prospective, observational study during their routine antenatal clinic visit and 282 nonpregnant subjects served as controls. A questionnaire was completed by the subjects to record their personal health history, family history of thyroid disease, and consumption of estrogen or antithyroid drugs. Thyroid palpation was performed to exclude the thyroid goiter. Thyroid function and urine iodine were measured by chemiluminescence and arsenic cerium analysis.The trimester-specific reference ranges in Nanjing were as follows: thyroid-stimulating hormone (TSH) 0.02 to 3.78 mIU/L, free thyroxine (FT4) 13.93 to 26.49 pmol/L, total thyroxine (TT4) 103.39 to 319.43 nmol/L in the 1st trimester. TSH 0.47 to 3.89 mIU/L, FT4 12.33 to 19.33 pmol/L, TT4 92.28 to 234.88 nmol/L in the 2nd trimester. TSH 0.55 to 4.91 mIU/L, FT4 11.38 to 19.21 pmol/L, TT4 83.54 to 258.12 nmol/L in the 3rd trimester. According to the TSH reference range recommended by American Thyroid Association (ATA), the prevalence of subclinical hypothyroidism, subclinical hyperthyroidism, hyperthyroidism, hypothyroxinemia, and thyroid peroxidase antibody-positive were 12.42%, 0.50%, 0.99%, 1.61%, and 11.80%, respectively, prevalence according to the trimester-specific reference range were 1.99%, 0.25%, 1.61%, 0.37%, and 1.61%, respectively, which showed elevated hypothyroxinemia incidence and declined incidence of subclinical hypothyroidism and hyperthyroidism.Trimester-specific reference range varied from that of ATA's recommendation, influencing the diagnosis, and treatment of pregnant thyroid disorders. To detect and control these disorders properly, setting up trimester-specific reference is clinically essential.