Objective Down syndrome (DS), also known as trisomy 21 syndrome, is a common and most harmful congenital chromosomal genetic disease. This study is aimed at exploring the effect of B-ultrasound NT scan in early pregnancy combined with serum screening in early and middle pregnancy for Down syndrome. Methods A total of 168 pregnant women who were diagnosed and treated in the obstetric clinic of our hospital from January 2019 to December 2021 were selected as the research objects. B-ultrasound NT scanning and serum detection in the early and middle trimester of pregnancy were performed, respectively. The accuracy of single detection and combined detection was analyzed and compared with the results of amniotic fluid cell chromosome examination as the gold standard. Results There were 4 cases of DS and 165 cases of non-DS. The serum PAPP-A, AFP, and UE levels in DS group were lower than those in non-DS group. β-HCG level and NT value were higher than those in non-DS group (all p < 0.05). Among 168 pregnant women, 5 cases were diagnosed as abnormal by ultrasonography, and 1 case was diagnosed as normal. By serological test, 20 cases with high risk of DS were diagnosed in 4 cases, and 148 cases with low risk of DS were diagnosed in 2 cases. Among 168 cases examined by serology combined with ultrasound, 10 cases with high risk of DS were found, and 4 cases were diagnosed; 158 cases had low risk of DS, and 0 cases were diagnosed. The negative predictive value, specificity, and coincidence rate of DS screening by the three methods were higher, and the positive predictive value and coincidence rate of combined screening were the highest (p < 0.05). The screening risk of Down syndrome was correlated with pregnancy outcome. The abnormal pregnancy rate in high-risk group was significantly higher than that in low-risk group, and the difference was statistically significant (p < 0.05). ROC curve showed that the sensitivity, specificity, and AUC of the combined detection were greater than those of serology and NT. Conclusion The application of B-ultrasound NT scan in early pregnancy combined with early and mid-term serum comprehensive screening in the screening of Down's infants is helpful to improve the diagnostic coincidence rate and reduce the occurrence of misdiagnosis.