Background: The reference intervals of thyroid hormone will change at different stages of pregnancy because of physiological alterations. On the other hand, the reference intervals of hyroid hormone will also change in different detection systems due to manufacturer’s methodology as well as different race. The objective in this study was to establish the assay method- and trimester-specific reference intervals for thyroid stimulating hormone, free thyroxine, and free triiodothyronine for pregnant women in the Chengdu.Methods: A prospective, population-based cohort study involved 23701 reference samples of pregnant women during the three trimesters and 8646 non-pregnant women with pre-pregnancy clinical and laboratory tests. The 2.5th and 97.5th percentiles were calculated as the reference intervals for thyroid stimulating hormone, free thyroxine, and free triiodothyronine at each trimester of pregnant women according to ATA Guidelines.Results: The reference interval of thyroid stimulating hormone in the 2.5th and 97.5th percentiles has a significant increasing trend from first trimester, to second trimester, and to third trimester, which was 0.08-3.79 mIU/L for first trimester, and 0.12-3.95 mIU/L for second trimester, and 0.38-4.18 mIU/L for third trimester, respectively (P < 0.001). However, the reference intervals of free thyroxine and free triiodothyronine in the 2.5th and 97.5th percentiles have significant decreasing trends from first trimester, to second trimester, and to third trimester, which were 11.87-18.83 pmol/L and 3.77-5.50 pmol/L for first trimester, and 11.22-18.19 pmol/L and 3.60-5.41 pmol/L for second trimester, and 10.19-17.42 pmol/L and 3.37-4.79 pmol/L for third trimester, respectively (both P < 0.001).Conclusion: It is necessary to establish assay method- and trimester-specific reference intervals for thyroid stimulating hormone, free thyroxine, and free triiodothyronine because the reference intervals of these thyroid hormones are significantly different at different stages of pregnancy.