Objective: To investigate the association of maternal serum biomarkers alpha fetal protein (AFP), β-human chorionic gonadotropin (HCG) and unconjugated estriol 3 (uE3) in the second trimester with fetal congenital heart disease (CHD) in low risk populations during the screening of Down's syndrome. Methods: 109 cases diagnosed with fetal CHD in the second trimester by fetal echocardiography were enrolled as the CHD group. Pregnancy- and gestational-age matched 344 cases without fetal CHD were used as the control group. The values of maternal serum biomarkers HCG, AFP and uE3 were tested and the association with CHD was analyzed. Results: The means of HCG multiple of median (MoM) and AFP MoM were higher, while the mean of uE3 MoM was lower in the CHD group than those of the control group (p < 0.05). The number of cases with HCG MoM ≥85% quantile, AFP MoM ≥85% quantile was more, while that with uE3 MoM ≤15% quantile was less in the CHD group than that of the control group (p < 0.05). The univariate logistic regression analysis showed that fetal CHD was associated with high values of HCG MoM and AFP MoM and low value of uE3 MoM as well as the HCG MoM ≥85% quantile, AFP MoM ≥85% quantile and the uE3 MoM ≤15% quantile. The multivariate logistic regression analysis showed that HCG MoM ≥85% quantile and AFP MoM ≥85% quantile or the uE3 ≤15% quantile were the independent factors of fetal CHD. In addition, the risk of fetal CHD was higher when one situation existed among the HCG MoM ≥85% quantile, AFP MoM ≥85% quantile and the uE3 MoM ≤15% quantile. The risk of fetal CHD was much higher when both the HCG MoM ≥85% quantile and AFP MoM ≥85% quantile existed or both the HCG MoM ≥85% quantile and the uE3 MoM existed ≤15% quantile. Conclusions: Second trimester maternal serum biomarkers may be useful indicators for fetal evaluation for CHD to screen positive pregnancies without identified chromosomal defects.