To eliminate the miscarriage risks caused by traditional invasive sampling methods, we develop a noninvasive prenatal paternity testing (NIPPT) method and evaluate its efficiency, reliability and sensitivity based on a scaled trial.We use maternal cell-free DNA and massive parallel sequencing to obtain NIPPT genotypes for parents and fetuses based on quality-controlled genome-wide single nucleotide polymorphisms (SNPs). In a preliminary testing, data from 14 pregnant women and 7 negative controls are used for setting threshold of fetal genotyping in reference to postpartum children. After that, those from 349 cases with pregnancies of 6-35 gestational weeks (GW) and 9 negative controls from non-pregnant women who have fertility experience previously are in-depth evaluated.In all cases, the biological fathers have been successfully identified from unrelated with a combined paternity index (CPI) of 3.58 × 10 - 1.46 × 10 for the cases versus 1.52 × 10 - 2.30 × 10 for the controls. For negative controls, fetal SNPs originating from previous pregnancies could not be detected. Our NIPPT results completely aligned with the invasive prenatal test results using PCR-CE STR methods.NIPPT can be applied to determine paternity accurately from 6 weeks after conception until birth and may serve as an alternative prenatal paternity test advantageous to the currently-used methods.