ABO is the most clinically important blood group system in transfusion and transplantation medicine. The popular ABO genotyping methods, such as the sequencing of exons 6 and 7 and sequence-specific primer (SSP)-PCR, often lead to ambiguous typing results. Long PCR-sequencing method was designed to analyze two regulatory regions (promoter and CBF/NF-Y enhancer regions) and all genomic sequences (except for intron 1) of the ABO gene. Using rapid DNA polymerase with high-fidelity, we amplified 6.3 kb and 7.3 kb for sequencing of enhancer-exon 1 and exons 2-7, respectively. ABO genotyping was performed using this technique in the peripheral blood of three unrelated families. The time requirements of the PCR amplification and purification processes were about 2.0 hours and 15 minutes, respectively. Five different ABO alleles (ABO A102, ABO A105, ABO O01, ABO O02, and ABO B101) with allele-specific CBF/NF-Y minisatellite repeats from three families were analyzed. All genotyping results agreed with serologic findings and results expected by Mendelian inheritance. Compared to conventional PCR-direct sequencing for ABO genotyping, this method proves simple and fast for the analysis of ABO genotypes. Therefore, it might be valuable in clinical transfusion or forensic applications.