Objective To explore the clinical significance of serological screening for pregnant women with high risk. Methods A total of 40046 cases of pregnant women during pregnancy were detected by time-resolved immunofluorescence in the serum AFP, concentration of beta HCG, and relevant assessment software was used to calculate the fetus with the 21-trisomy syndrome, 18-trisomy syndrome and open neural tube defects (ONTD) risk of value. Prenatal screening of pregnant women with different age, gestational age and weight was analyzed. Results A total of 2298 high-risk pregnant women were screened, with a positive rate of 5.74%. Among them, 2165 cases had high risk of 21- trisomy syndrome with a positive rate of 5.41%, high risk of 18-trisomic syndrome with 0.04% positive rate. 133 cases had high risk of NTD with 0.33% positive rate. Different gestational weeks was a factor for 21-trisomic syndrome, a high risk ratio were founded at 15 weeks (15-15⁺⁶ weeks), with a significant difference (P<0.05). There was no significant difference in the risk of the three diseases among women with different weight (P>0.05).The higher the age, the higher the risk ratio of 21-trisomic syndrome and open neural tube malformation, the difference was statistically significant (P<0.05). Conclusion Maternal serological screening during the middle pregnancy is of great significance for early detection of high risk pregnant women. Taking full advantage of the screening results and taking appropriate interventions as early as possible can reduce the risks of 21-trisomy syndrome, 18-trisomy syndrome and open neural tube malformation in the fetus.