The objective of this study is to establish self-sequential longitudinal reference intervals of thyroid function in normal pregnant women. According to the selection criteria, 301 cases were taken as the normal pregnant population to establish a normal reference range. Meanwhile, 150 healthy women were selected as the normal non-pregnant control group. To establish their own self-sequential longitudinal reference intervals, we collected samples five times in every case throughout the gestation (including first trimester, second trimester, third trimester, prenatal and postpartum), and detected the levels of thyroid stimulating hormone (TSH), free thyroxine (FT4), thyroid peroxidase antibodies (TPO-Ab), and then established the self-sequential longitudinal reference intervals. The levels of TSH, FT4 and TPO-Ab were quantified by electrochemistry immunoassay (ECL) and statistically analyzed using SPSS 13.0 software. Serum TSH of normal pregnant women was at a low level in the first trimester (P < 0.05) and began to rise continuously. Not until prenatal phase was it restored to the non-pregnant state (P > 0.05). During pregnancy, serum FT4 of normal pregnant women were consistently lower than non-pregnant levels (P < 0.05) and kept at low levels. Serum TPO-Ab increased significantly in the third trimester and prenatal phase (P < 0.05). Of normal pregnant women, 6.5% were TPO-Ab positive. In conclusion, the reference intervals in our case will reflect the changes of thyroid function in pregnant women more realistically, resulting in a more accurate value for clinical diagnosis and therapy.