Objective: To explore the application of fragmental enrichment method of fetal free DNA based on carboxylated nano magnetic beads combined with high-throughput sequencing technology in non-invasive prenatal testing. Methods: A total of 20,868 pregnant women who performed non-invasive prenatal diagnosis in the prenatal diagnosis center of our hospital from August 2019 to August 2021 were enrolled. The differences between traditional purified DNA extraction method and fragmental enrichment method combined with high-throughput sequencing technology on the quality of extracted DNA and the diagnosis of fetal chromosomal abnormalities were compared. Results: A total of 5,914 pregnant women were detected by traditional purified DNA extraction method combined with high-throughput sequencing, and 33 of them (0.56%) needed to be retested due to low concentration of circulating DNA; While 14,954 pregnant women were detected by fragmental enrichment method combined with high-throughput sequencing, 2 cases (0.01%) needs repeat blood sampling for examination, and there was a significant difference in the proportion of repeated blood sampling between the two groups (0.56% vs.0.01%, p＜0.001). Compared with the traditional DNA extraction method (23/5,914, 0.39%), the fragmental enrichment method combined with high-throughput sequencing (47/14,954, 0.31%) showed no significant difference in the chromosome abnormality rate (trisomy 21+trisomy 18+trisomy 13) (p＞0.05). After amniocentesis and postpartum follow-up review, there was 1 case missed in each group, and there was no significant difference between the two groups (0.02% vs.0.007%, p＞0.05). Particularly，compared with the traditional DNA purification method, the fragmental enrichment method showed no significant difference in the positive rate of trisomy 21 (54.55% vs.64.86% p＞0.05), trisomy 18 (83.33% vs.60% p＞0.05) and trisomy 13 (50.00% vs.25.00% p＞0.05) in maternal peripheral blood. Conclusions: The fragmental enrichment method of fetal free DNA based on carboxylated nano magnetic beads can enrich the fetal free DNA content in maternal peripheral blood circulation, thereby reducing the risk of repeated blood drawing in pregnant women. In addition, the fragmental enrichment method combined with high-throughput sequencing will not lead to the loss of target DNA, reducing the detection rate of chromosomal abnormalities.