OBJECTIVE To construct eukaryotic expression vectors for human ABO and subgroup genes A101, B101, CisAB01, Ael05, B(A)04 and Bw03, and validate their expression in vitro. METHODS Total RNA was isolated from individuals with the A101 and B101 subgroups. cDNA of A101 and B101 was synthesized by reverse transcription and amplified with specific primers. Subgroup genes CisAB01, Ael05, B(A)04 and Bw03 were then amplified with PCR for site-directed mutagenesis. Fragments of the ABO genes were directionally linked to pcDNA3.1 positive-eukaryotie expression vectors. After antibiotic screening, the sequences were analyzed. The vectors were transfected into Hela cells, and the expression of target proteins was detected by Western blotting and immunofluorescence assay. RESULTS Sanger sequencing has confirmed that pDNA3.1-A101, pDNA3.1-CisAB01, pDNA3.1-Ael05, pDNA3.1-B101, pDNA3.1- B(A)04, pDNA3.1-Bw03 positive-eukaryotic expression vector were successfully constructed. The results of Western blotting and immunofluorescence revealed clear presence of the expressed proteins. CONCLUSION Eukaryotic expression vectors for ABO subgroup genes were successfully constructed and worked well in Hela cells in vitro, which can facilitate further study of the ABO blood group proteins.