The study analyzed indications for prenatal diagnosis in 3,931 pregnant women (3,952 amniotic fluid samples) in three groups, with ultrasound anomalies being the main indications for copy number variation detection. Sequencing of copy number variations (CNV-Seq) has become a first-line technology for prenatal diagnosis. The detection rate of chromosomal abnormalities was 3.72% for karyotyping and 16.20% for CNV-Seq. When combining CNV-Seq with karyotyping, the detection rate increased from 7.02% to 15.81%. CNV-Seq confirmed the small supernumerary marker chromosome and unidentified karyotypes detected by karyotyping, both methods revealing low-level mosaicism. In the era of CNV-Seq, the target population for prenatal diagnosis is expanding. The combination of CNV-Seq with karyotyping is an effective and relatively cost-effective strategy for prenatal diagnosis.