Originally prenatal diagnosis was confined to the diagnosis of chromosome abnormalities by cultured cells in amniotic fluid. In recently, cell-free fetal nucleic acids in the supernatant of amniotic fluid, which is usually discarded, can be used as a sample for prenatal diagnosis and monitor the ongoing development of human fetuses in vivo. Amniotic fluid supernatant is a valuable and widely available but under-utilized biological resource. It opened a new way to insights fetal development and pathology. The review was to summarize the current knowledge on cell-free fetal nucleic acids in amniotic fluid.