The purposes of this study were to use quantitative three-dimensional (3-D) ultrasonography to establish the normal charts of three fetal ear-growth indexes, (ear length, ear width and ear area), and to validate their efficacy in the prenatal diagnosis of fetal trisomies, either separately or as a combination. Using quantitative 3-D ultrasonography, we performed a prospective study to measure the three fetal ear growth indexes (i.e., ear length, ear width and ear area) in 129 singleton pregnancies, including 122 normal fetuses and 7 trisomies. The ear shape was also evaluated in these fetuses using both 2-D and 3-D ultrasonography. Our results showed that: First, 3-D ultrasonography offers better visualization and easier evaluation of fetal ears than 2-D ultrasonography. Second, when using the quantitative assessment of 3-D ultrasonography, the measurements of ear length, ear width and ear area were all correlated significantly with gestational age in normal pregnancies (r = 0.881, 0.848, and 0.890, respectively). In addition, 3 of 7 fetal trisomies had ear length below the tenth percentile, 1 had ear width below the tenth percentile, and 2 had ear area below the tenth percentile. Furthermore, with the combination of these three measurements, (including ear length, ear width and ear area), the sensitivity in detecting fetal trisomies was elevated to 57.1% and the specificity was 83.2%. In conclusion, 3-D ultrasonography reduces the limitations of 2-D ultrasonography in the evaluation of fetal ears. However, fetal ear measurement may not be used as a single ultrasonographic parameter in identifying aneuploid fetuses. We recommend using the combination of these three ear growth indexes to enhance the detection rate of aneuploidy.