Abstract: Objective To analyze serum prolactin (PRL) levels during pregnancy in healthy single pregnant women of childbearing age in Suzhou, and to establish and verify the reference interval of serum PRL. Methods From January to March, 2022 , the data of pregnant women with healthy single pregnancy at childbearing age were collected and prospectively followed up until delivery. According to the gestational age, the subjects were divided into early pregnancy group (less than 14 weeks), middle pregnancy group (14-27⁺⁶ weeks) and late pregnancy group (≥28 weeks). PRL was determined by Soling LIAISON XL automatic chemiluminescence immunoassay and LIAISON^® prolactin. After eliminating outliers, the medical reference interval of serum PRL during pregnancy was established by percentile method (P_2.5~P_97.5). In addition, 20 samples of healthy single pregnant women of childbearing age were randomly collected in early, middle and late pregnancy to verify the established reference interval. When no more than two PRL measurements in each group exceeded the established reference interval, they were considered validated. Results A total of 170 participants were included in the early pregnancy group, 229 participants in the middle pregnancy group and 130 participants in the late pregnancy group. There are significant differences in serum PRL levels in pregnant women at different stages of pregnancy. With the increase of gestational age, its level increased. The reference intervals of serum PRL in early, middle and late pregnancy were 477~4270 mIU/L, 1060~6574 mIU/L and 3497~18274 mIU/L, respectively. All the established reference intervals have been verified. Conclusions This study established the reference interval of serum PRL during pregnancy of healthy single pregnant women of childbearing age in Suzhou area, to provide help for clinical rational application of this index, and further provide reference for the prevention of pregnancy-related diseases.