Background: Congenital heart diseases, as the most common congenital anomaly, are a major cause of serious morbidity and mortality. Objectives: This study aimed to evaluate the level of alpha-fetoprotein in the second trimester of pregnancy and its association with congenital heart defects. Methods: Following a cross-sectional design, singleton pregnant women in the second-trimester were enrolled for the α-fetoprotein (AFP) screening test. Those with normal screening results at the first and second tests were assigned to the control group. Mothers with nuchal translucency (NT) screening equal or greater than 3% of the 99th percentile, without chromosomal anomalies, confirmed with amniocentesis, were referred for a fetal echocardiogram and in presence of congenital heart defects symptoms, assigned to the intervention group. The study groups were compared concerning the serum AFP level and maternal and neonatal outcomes in the second trimester of pregnancy. Statistical significance was considered when P-value < 0.05. Results: A total of 270 women were enrolled in the study, with a mean AFP level of 1.868 ± 0.87 and 1.374 ± 0.39 in the case and control groups, respectively. The mean level of AFP was significantly different between the two groups (P < 0.001). In the second trimester, an AFP level lower than 1 was associated with a significant increase in the likelihood of developing congenital heart anomalies in the fetus. Conclusions: This study demonstrated that the value of maternal AFP level in the second trimester can be considered as an appropriate screening test to predict the incidence of congenital heart defects in neonates.