Background: Thyroid disorders are common during pregnancy. To date, a limited number of studies have reported differences in serum thyroid hormone concentrations between different ethnic groups. We sought to establish gestational age-specific reference intervals for serum levels of thyroid hormones in a multi-ethnic population and investigate whether separate reference intervals should be used for different ethnic groups. Methods: A total of 926 pregnant women from multiple ethnic groups attended four separate study visits spanning the three trimesters. Venous blood samples were taken at 9 to 14 weeks, 18 to 22 weeks, 28 to 32 weeks, and 34 to 39 weeks of gestation. Serum concentrations of thyroid-stimulating hormone (TSH), free thyroxine (T4), free triiodothyronine (T3), total T4, total T3, thyroid peroxidase antibody and thyroglobulin antibody were measured using Abbott Architect immunoassays. A total of 562 women with singleton pregnancies were found to be negative for both thyroid autoantibodies at all four study visits and thus included in the reference sample group for the establishment of reference intervals (2.5th to 97.5th percentiles). Results: Reference intervals for serum thyroid hormones at 9–14 weeks of gestation derived from the combined group of pregnant women are as follows: TSH, 0.01–2.39 mIU/L; free T4, 11.4–19.5 pmol/L; free T3, 4.23–6.69 pmol/L; total T4, 77.8–182.4 nmol/L; total T3, 1.39–2.97 nmol/L. No differences in the five thyroid parameters’ reference intervals are detectable among the ethnic groups except that at study visit 3 (28–32 weeks of gestation), the upper reference limit of total T3 in Malays (3.20 nmol/L; 90% CI, 2.99–3.76 nmol/L) is slightly higher than that in Chinese (2.86 nmol/L; 90% CI, 2.70–2.98 nmol/L). Conclusions: The findings from this study on a multi-ethnic cohort highlight the importance of establishing locally derived and gestational age-specific reference intervals for the five thyroid hormone parameters.