To develop and validate a novel 6-dye STR(short tandem repeat) 25-plex DNA typing system for forensic DNA profiling and databases. In this study, a novel STR 25-plex DNA typing system that includes 24 autosomal STRs (D1S1656, D2S1338, D2S441, D3S1358, D5S818, D6S1043, D7S820, D8S1179, D10S1248, D12S391, D13S317, D16S539, D18S51, D19S433, D21S11, D22S1045, CSF1PO, FGA, Penta D, Penta E, TH01, TPOX, vWA, D11S4463) and Amelogenin was developed. Validation studies demonstrated the sensitivity, accuracy, and reproducibility of our novel STR 25-plex DNA typing system. The sensitivity of the STR 25-plex DNA typing system was demonstrated by the ability to obtain complete profiles from as little as 0.125ng of human DNA. Specificity testing was demonstrated by the lack of cross-reactivity to a variety of commonly encountered animal species and microbial pool. For stability testing, full profiles were obtained with humic acid concentration ≤60ng/μL and hematin ≤600μM. For forensic evaluation, the selected 24 autosomal STRs followed the Hardy–Weinberg equilibrium. Since 24 autosomal STRs were independent from one another, PM (Probability matching) was 3.5434×10-28, TDP (Total Probability of Discrimination Power) was 0.999999999999999999999999969863, and CEP (Cumulative probability of exclusion) was 0.99999999375. The new STR 25-plex typing system is sensitive, reproducible, and stable, therefore it is highly applicable for use in national DNA database and can help to facilitate international data sharing.