Different newborn screening (NBS) programs have been practiced in many countries since the 1960s. It is of considerable interest whether next-generation sequencing is applicable in NBS. We have developed a panel of 465 causative genes for 596 early-onset, relatively high incidence, and potentially actionable severe inherited diseases in our Newborn Screening with Targeted Sequencing (NESTS) program to screen 11,484 babies in 8 Women and Children's hospitals nationwide in China retrospectively. The positive rate from preliminary screening of NESTS was 7.85% (902/11,484). With 45.89% (414/902) follow-up of preliminary positive cases, the overall clinically confirmative diagnosis rate of monogenic disorders was 12.07% (50/414), estimating an average of 0.95% (7.85% × 12.07%) clinical diagnosis rate, suggesting that monogenic disorders account for a considerable proportion of birth defects. The disease/gene spectrum varied in different regions of China. NESTS was implemented in a hospital by screening 3923 newborns to evaluate its clinical application. The turn-around time of a primary report, including the sequencing period of < 7 days, was within 11 days by our automatic interpretation pipeline. Our results suggest that NESTS is feasible and cost-effective as a first-tier NBS program, which will change the status of current clinical practice of NBS in China.