Bacterial artificial chromosome (BAC) marker-microsphere identification/separation technique [BACs-on-Beads (BoBs)] not only has a high detection rate for major chromosomal changes, but also for the other 9 microdeletion syndromes. In this study, the application value of BoBs combined with karyotype detection in prenatal diagnosis was evaluated. The amniotic fluid samples of 132 pregnant women with prenatal diagnosis indications in Harbin Red Cross Central Hospital from June 2018 to June 2019 were collected and subjected to the detection of BoBs and routine karyotyping. Among the 132 pregnant women's amniotic fluid samples, 30 cases were abnormal in BoBs detection, with a detection rate of 22.73%, and 29 cases were abnormal in chromosome karyotype analysis, with a detection rate of 21.97%. Among them, 1 case of DiGeorge Type I microdeletion syndrome BoBs was successfully detected. The karyotype analysis failed to detect the same syndrome; the total coincidence rate of two methods was 99.24%, the positive coincidence rate was 100.00%, and the negative coincidence rate was 99.03%; the sensitivity, specificity and positive predictive value (PPV), and negative predictive value (NPV) of the chromosome karyotype analysis was 96.67%, 100%, and 99.03%, respectively; the accuracy, specificity, and PPV/NPV of BoBs detection were 100%. When BoBs technology is combined with chromosome karyotype analysis, it can increase the detection rate of fetal chromosomal abnormalities, which could provide a basis for clinical prevention and follow-up diagnosis and treatment.