We aimed to develop a new method of screening for Klinefelter Syndrome by detecting amniotic fluid punctures to complement the current methods. Two commercially available genomic DNA extracted from the amniotic fluid puncture of the pregnant woman with the Klinefelter syndrome fetus, two genomic DNAs extracted from two healthy females and four genomic DNAs extracted from four healthy males were used as the qPCR template DNAs and the commercially available Sybr green qPCR master mix were used; we designed and synthesized 5 pairs of qPCR primers respectively corresponding to IL-10 gene on 1# chromosome, STAT1 gene on 2# chromosome, CXCR3 gene on X chromosome, TSPY1 gene on Y chromosome and LINC00458 on 13# chromosome. We then performed Sybr green qPCR measurement. We processed the qPCR data by mathematical calculation and finally formed a new algorithm. Using the new algorithm, we easily distinguished the Klinefelter syndrome samples out of the normal male samples. We developed a new method of screening for Klinefelter syndrome for the male fetus by detecting amniotic fluid punctures to complement the current methods.