This study explored the clinical application of karyotype analysis combined with BACs-on-Beads (BoBs) technology in prenatal diagnosis. A total of 558 pregnant women who were admitted to Xuzhou Maternity and Child Health Care Hospital from July 2015 to June 2017 were enrolled in this study. All the subjects underwent amniocentesis. BoBs assay was performed for subjects in the observation group, and karyotype analysis was performed for subjects in the control group. The main technical indicators of subjects in the two groups were summarized, and cases of chromosome abnormalities were further evaluated. Clinical follow-up of their pregnancy and neonatal birth was undertaken. Finally, the chromosomal manifestations of these patients were compared with those of normal male and normal female, as well as common chromosomal abnormalities. All 558 pregnant women underwent amniocentesis again. Karyotype analysis combined with BoBs assay of amniotic fluid was performed. Cases of chromosomal abnormalities detected were: 75 cases of trisomy 21, 20 cases of trisomy 18, 1 case of trisomy 13, 27 cases of sex chromosomal abnormalities, 12 cases of balanced chromosome translocation, and 2 cases of chromosome microdeletion. The results indicated that karyotype analysis combined with BoBs technology for prenatal diagnosis was easy to perform, and provided quick results with high accuracy. The two testing methods were complementary to each other, which significantly improved the diagnostic rate of chromosomal abnormalities thus reducing birth defects and guiding continued pregnancy of high-risk pregnant women.