Objective: To explore whether CNV-seq can be used as a first-tier diagnostic method or even replace karyotyping for prenatal diagnosis alone. Method: A retrospective study was conducted to 4230 amniocentesis samples with CNV-seq detection and G-banding karyotyping simultaneously. The indications for prenatal diagnosis included abnormal result on Down’s Syndrome Screening, abnormal fetal ultrasound, abnormal result on noninvasive prenatal screening, and so on. The results that lead to birth defects definitely were defined as abnormalities, which included aneuploidy, mosaic aneuploidy, large deletion/duplication and pathogenic copy number variations (pCNVs). Results: 278 cases of abnormalities was identified by karyotyping with an abnormal detection rate of 6.69%(283/4230). In addition, for all the abnormalities identified by karyotyping, CNV-seq also identified another 58 cases of abnormalities. A total of 341 cases of abnormalities were identified by CNV-seq with an abnormal detection rate of 8.06%(341/4230), higher than that of karyotyping. Abnormal detection rate of CNV-seq for the groups with abnormal result on noninvasive prenatal screening, abnormal fetal ultrasound, abnormal parental chromosome, adverse pregnancy history, abnormal result on Down’s Syndrome Screening, volunory testing and advanced maternal age were increased by 2.53%, 1.91%, 1.44%, 1.24%, 1.01%, 0.99% and 0.62% over the karyotyping respectively. Conclusion: CNV-seq and karyotyping had the same effectiveness in identifying aneuploidies, but CNV-seq had absolute superiority in the detection of low proportion of mosaics, imbalanced structural abnormalities. This prevents the birth of fetuses with these chromosome abnormalities that cannot be identified by karyotyping. CNV-seq can replace karyotyping in prenatal diagnosis for chromosome test alone.