Abstract: Objective: To establish a new method for non-invasive prenatal diagnosis. Methods: A single-copy ZFY gene of Y-chromosome of fetal DNA was amplified by nested polymerase chain reaction （PCR） from 46 pregnant maternal plasma （10-40 weeks）. A 354 bp and 307 bp specific fragment were obtained. The maternal plasma samples of 46 pregnant women were used directly for nested PCR. Results: The fragment was identified in 32 of 39 pregnant women plasma bearing male fetuses. The diagnostic rate was 82.1%（32/39）, 24 of 32 women have positive band in both amplifications, 8 of 32 women have positive band at the second amplification. The positive rate was increased by nested PCR （from 61.5% to 82.1%）. The total accuracy was 84.8%（39/46）. Conclusion: The circulating fetal DNA in maternal plasma may have been detected by nested PCR which have high sensitivity and specificity for the clinical application.