Abstract: Background：It is unclear about the effect of the rate of folic acid supplementation and unhealthy life habits in childbearing couples on the level of serum. Objective：To describe the serum folate levels in childbearing couples in Kunshan City and investigate the associations of tobacco exposure and alcohol drinking with serum folate levels. Design：Crosssectional study. Methods：The couples with the age of wife below 45 were enrolled from the premarital or prepregnancy examination clinic in Maternal and Child Health Center in Kunshan City. The questionnaires and blood samples were collected to analyze the relationship of the tobacco exposure defined as smoking or being passively exposed to tobacco smoke in the past 3 months and alcohol drinking defined as drinking alcoholic drink for at least one time in the past 30 days with serum folate levels. As there is no standard reference levels of serum folate for the childbearing couples, we used the P50 of serum folate level in this study to define the low folate group(<P50) and normal group (≥P50) to ensure a maximum statistic power. The covariates included age, sex, BMI, education, and the use of folic acid supplementation. Main outcome measures：Serum folate levels. Results：A total of 3 990 couples were enrolled from April 2019 to June 2021. The mean age were 28.5±3.8 and 27.4±3.5 years for the males and females. The prevalence of tobacco exposure, drinking and folic acid supplementation before pregnancy for males and females were 31.3% and 0.9%, 27.9% and 6.0%, 0.7% and 2.7%, respectively. Median serum folate levels in fathers and mothers were 12.7 ng·mL-1 (14.0, 26.1) and 18.8 ng·mL-1 (9.5, 17.0) correspondingly. After adjustment for covariates, the tobacco smoke exposure was associated with higher odds of low serum folate in whole population (OR=1.48, 95% CI:1.281.72), and this association was further observed in males (OR=1.46, 95%CI: 1.261.71), but not in females. Alcohol drinking was significantly associated with low serum folate levels in the whole population (OR=1.24，95%CI：1.091.42),males (OR=1.21，95%CI：1.051.42),and females (OR=1.34，95%CI：1.021.76). Conclusion：Tobacco exposure and alcohol drinking before pregnancy were associated with low serum folate, which may exist gender difference.

Key words: Serum folate, Tobacco exposure, Alcohol drinking, Child-bearing couples