To explore the application value of serum inhibin A (INH-A), alpha-fetoprotein (AFP), unconjugated estriol (uE3), and β-human chorionic gonadotropin (β-hCG) levels combined with non-invasive prenatal testing (NIPT) for screening Down’s syndrome (DS) during the second trimester of pregnancy. Methods: The clinical data of 2,058 pregnant women between March 2017 and August 2020A were selected retrospectively. All these women had undergone serological triple screening (the detections of β-hCG, AFP, and uE3) and INH-A test. The women with high-risk of fetal DS by serological screening were further given NIPT. The results of amniocentesis were as the golden standard, and the diagnostic efficiency of different screening methods for DS was analyzed. Results: According to the results of amniocentesis, all these women were divided into group A1 (16 women with fetal DS) and group A2 (2,042 women without fetal DS). The levels of serum AFP, β-hCG, and INH-A of the women in group A1 were significantly higher than those of the women in group A2, while the uE3 level of the women in group A1 was significantly lower (P＜0.05). There were 9 women with high risk of fetal DS by serological triple screening, 10 women with high risk of fetal DS by serological triple screening and INH-A level, and 15 women with high risk of fetal DS detected by serological triple screening and INH-A level combined with NIPT. The false positive rate of serological triple screening and INH-A level combined with NIPT for screening DS was the lowest (0.49%). The sensitivity, the specificity, the accuracy, and the positive predictive value of serological triple screening and INH-A level combined with NIPT for screening DS were 93.8%, 99.5%, 99.5%, and 60.0%, which were significantly higher than those of serological triple screening and those of serological triple screening and INH-A level (all P＜0.05). Conclusion: The efficiency of the levels of serum β-hCG, AFP, uE3, and INH-A combined with NIPT for screening fetal DS of the pregnanat women during the second trimester of pregnancy is the best, which has higher diagnostic efficiency.