Human identification and paternity testing are usually based on the study of STRs depending on their particular characteristics in the forensic investigation. In this paper, we developed a sensitive genotyping system, SiFaSTR((TM)) 23-plex, which is able to characterize 18 expanded Combined DNA Index System STRs (D3S1358, D5S818, D2S1338, TPOX, CSF1PO, TH01, vWA, D7S820, D21S11, D10S1248, D8S1179, D1S1656, D18S51, D12S391, D19S433, D16S539, D13S317, and FGA), three highly polymorphic STRs among Chinese people (Penta D, Penta E, and D6S1043), one Y-chromosome Indel and amelogenin using a multiplex PCR; the PCR amplified products were analyzed using the Applied Biosystems 3500 Genetic Analyzer. Full genotyping profiles were obtained using only 31.25 pg of control DNA; various case-type specimens, as well as ten-year-old samples were also successfully genotyped. Additionally, in the validation studies, this multiplex was demonstrated to be human-specific and compatible with the interference of multiple PCR inhibitors. The system also enabled the detection of mixtures, and complete profiles could be obtained at the mixed ratios of 1:1, 1:3, and 3:1. The development and validation study here illustrated that the SiFaSTR((TM)) 23-plex system is accurate, powerful, and more sensitive than many other systems. What's more, the population data in our study not only illustrated that this 23-plex typing system was straightforward and efficient but also expanded the Chinese STR database, which could facilitate the appropriate application of the 23 genetic markers in forensic genetics, especially in the Chinese population.