Abstract： Objective To establish the genetic test technique of trisomy 21 concurrently conducts with prenatal diagnosis for hereditary hearing loss. Methods Fifty-four pregnant women who underwent prenatal diagnosis for hearing loss of their fetuses in Chinese People's Liberation Army General Hospital from March 2009 to May 2010 were enrolled in this study. All probands from the deaf families have confirmed the causative mutation for hearing loss in Genetic Testing Center in Chinese People's Liberation Army General Hospital. The mean age of 54 pregnant women is 31 years at pregnancy of 18 - 26 weeks, 5 cases ＞ pregnancy of 23 weeks, 9 cases ≥ 35 years. All subjects did not conduct the serologic tests for trisomy 21before. Fifteen to twenty ml amniotic fluid was drawn from 49 cases at pregnancy of 18 - 23 weeks and 5 cases ＞ pregnancy of 23 weeks. One to two ml umbilical blood was drawn from 5 cases ＞ pregnancy of 23 weeks. For 9 cases ≥ 35 years, amniotic fluid cell culture and karyotyping analysis were conducted concurrently. A multiple quantitative fluorescent ( QF) PCR and six microsatellite markers were applied to as trisomy 21. Results (1) Fifty-four fetuses were successfully conducted prenatal genetic diagnosis for hearing loss (included GJB2 and SLC26A4). Ten fetuses copied the exactly same genotypes as the probands. The other 44 cases fetuses did not copy the same genotypes as the probands and won't develop hearing loss. The hearing test showed normal hearing for the neonates. (2) All the 54 fetuses were excluded of trisomy 21 by QF-PCR and were verified after birth. Five fetuses with advanced maternal age were performed karyotyping analysis and showed normal. The diagnostic results of QF-PCR can be obtained in 1 - 3 days without misdiagnosed. Conclusions QF-PCR is an efficient, rapid and accurate technique for detection of trisomy 21 without increasing sample amount. It can be used for fetuses who were undertaken hearing loss gene test or other prenatal gene test.