The aim of study was to investigate the construction, identification and expression of human B7.1 (CD80) eukaryotic expressing vector on HL-60 cells. B7.1 gene was subcloned from the cloning vector using PCR. The PCR products and eukaryotic expressing vector (pHook) both were separately digested with ApaI, SalI and were ligated using T4 DNA ligase. The ligases products were transduced into DH-5alpha. B7.1 gene containing clones was selected by digestion with ApaI and SalI, and were further confirmed by sequencing of DNA. HL-60 cells were transfected with B7.1 by using lipofectamine and detected by immunofluorescence, SABC and FACS methods. The results showed that the size of PCR products was about 620 bp. Five clones were ampicillin-resistant and all could be digested by ApaI and SalI to produce 620 bp gene fragment that had the same size of B7.1, which means that the B7.1 recombinant vector has been constructed successfully. Further sequencing confirmed the validity of the construction. No nucleotide mutation was found, B7.1 effectively expressed on HL-60 cells with 70%, 65% and 92.7% by immunofluorescence, SABC and FACS respectively. It is concluded that the human B7.1 (CD80) eukaryotic expressing vector can be successfully constructed by molecular cloned methods and can stably effectively express on the membrane of B7.1-negative acute myelocytic leukemia (AML) cell line HL-60. It is inferred that the vaccine prepared by using this method may have immunotherapeutic and immuno-protective effects.