Objective To compare and evaluate different DNA extraction methods for batches of bloodstain samples during the establishment of national DNA database. MethodsDNA was extracted from bloodstain samples collected for the establishment of DNA database using manual Chelex-100, Biomek 3000 automation workstation with Chelex-100 and DNA-IQTM magnetic bead method respectively, and the quantity of DNA was determined by the real-time fluorescent quantitative PCR technique. Genotyping was performed using the AmpFLSTR STR Identifiler kit and 3100 genetic analyzer. ResultsThe concentrations of DNA templates obtained by manual Chelex-100, automatic Chelex-100 and DNA-IQTM magnetic bead method were 0.593?0.131ng/?l, 0.579?0.096ng/?l and 0.447?0.056ng/?l respectively, and the success rates of genotyping with these DNA templates were 100%, 98.9% and 99.5% respectively. ConclusionThe automatic Chelex-100 method proposed by this study can be applied in the establishment of large-scale DNA databases.