Newborn screening (NBS) is an effective way for 3-step prevention of birth defects. The suitable technology and rational NBS screening diseases are critical for each country and area. High-throughput sequencing has shown high application potential in NBS. However, lack of sequencing strategy for monogenic inherited diseases NBS in China. In this study, we systematically evaluated the application efficiency of different sequencing approaches for NBS, and a gene-disease association list (NeoExome panel) for the Chinese population with 601 genes was designed based on the top rare disease list and databases. In the 1000 Genomes Project, 7.6% (23/301) were NGS positive. Among the 3249 neonates recruited, NGS positive rate was 12.0%. In the 200 conventional NBS (+) subgroup, 118 were NGS positive, with 76.3% (90/118) neonates harboring consistent results of conventional NBS and NGS; in the conventional NBS (-) subgroup, the NGS positive rate was 8.9% (271/3049). Our study designed a personal NBS targeted-sequencing NeoExome panel of monogenic inherited diseases for Chinese, which has shown acceptable performance.