Background: To examine and evaluate the alterations in plasma folate, homocysteine (HCY), and erythrocyte folate (FOA) concentrations among expectant mothers following folic acid supplementation in distinct pregnancies. Methods: A retrospective analysis was conducted with 416 pregnant women, divided into two groups: an observation group (n=210) who consistently took folic acid throughout pregnancy, and a control group (n=206) who only supplemented during early pregnancy. Key outcomes included plasma folate, HCY, and FOA levels, as well as pregnancy complications and neonatal outcomes. Results: Plasma folate levels were significantly higher in the observation group (10.42 +/- 2.96 ng/mL) compared to the control group (7.51 +/- 1.58 ng/mL, P<0.001). HCY levels were lower in the observation group (6.54 +/- 1.51 mmol/L) versus the control group (10.58 +/- 1.27 mmol/L, P<0.001). FOA levels were also higher in the observation group (486.42 +/- 105.29 ng/mL) compared to the control (430.20 +/- 75.12 ng/mL, P<0.001). The observation group had reduced rates of cesarean section (26.19% vs. 36.41%, P=0.025), anemia (3.81% vs. 8.74%, P=0.038), and hypertension during pregnancy (5.24% vs. 11.65%, P=0.018). Pearson correlation analysis showed a positive correlation between plasma folate and FOA (r=0.116, P<0.05) and negative correlations between plasma folate and HCY (r=-0.411, P<0.05) and between FOA and HCY (r=-0.286, P<0.05). The observation group had significantly lower FBG and P2BG levels and a reduced incidence of gestational anemia and HIP compared to the control group (P<0.05). Cesarean sections were also less frequent in the observation group. Newborns in the observation group had significantly greater height, weight, head circumference, and chest circumference than those in the control group (P<0.05). Conclusions: Consistent folic acid supplementation throughout pregnancy significantly improves maternal folate status, decreases pregnancy-related complications, and enhances neonatal health outcomes. These findings underscore the need for continuous folic acid intake during pregnancy, which could inform clinical guidelines and public health policies to optimize maternal and neonatal health.