In forensic investigations, ABO genotyping is often performed to obtain information for individual identification. This information is especially effective in the analysis of highly decomposed samples with which the serological method for ABO typing cannot be used due to ABH antigen deterioration. The authors previously reported on the development and improvement of a microarray-based method that can be applied for simultaneous ABO genotyping and human identification using two pairs of probes to detect major O or B allele-specific single nucleotide polymorphisms (SNPs) in the ABO gene and a probe to determine the human-specific D17Z1 sequence. In this study, a probe pair for B allele-specific SNPs was redesigned and selected because those used in the previous study appeared less than optimal in regard to specificity between BB and B heterozygous genotypes, which could give rise to false results. When DNA from bodily fluids (blood, saliva, semen and vaginal fluid), tissues and blood from cadavers, and blood stains were analyzed using the redesigned chip, the O and B index values, which reflect fluorescent patterns of each probe pair, could be clearly separated according to genotype. On the basis of these results, threshold values for each index were set and their use for genotype determination in analysis of aged stains and bone samples was verified. Finally, validation was performed with respect to the effect of the amount of input DNA for ABO genotyping by analyzing 0.0625 to 0.5 ng of DNA with the BO genotype. The results demonstrated that more than 0.25 ng of DNA is necessary for accurate genotyping with this method. Due to its simplicity, rapidity and accuracy, the approach is expected to be useful for ABO genotyping in forensic investigations.