Objective To select hepatic progenitor cell clones from human fetal liver cells (FLCs) based on α-fetoprotein (AFP) promoter activities. Methods AFP promoter was amplified from human fetal liver genome by polymerase chain reaction (PCR) and cloned into pGL3-basic vector, forming the pGL3-AFP in a direct orientation for the promoters to drive firefly luciferase expression. To analyze the specificity of the AFP promoter, the pGL3-AFP and pRL-TK plasmids were co-transfected into HepG2, A549, and HeLa cells; the latter two cells do not express AFP. In order to obtain the homogenous cell populations, the proliferating colonies were isolated from clonal cultures of FLCs, and the AFP promoter activity of each clone was analyzed in each well of the cell lysates after co-transfection of pGL3AFP and pRL-TK plasmids for 48hours. Immunofluorescence was used to confirm the AFP expression in each clone. Results The results of PCR, restriction enzyme cutting and DNA sequencing confirmed that the pGL3-AFP had been constructed successfully. After co-transfection of the pGL3-AFP and pRL-TK plasmids, the AFP promoter activity was detected in HepG2 cells, but was weakly detected in A549 and HeLa cells, indicating that the AFP promoter could drive downstream firefly luciferase gene expression in AFP-expressing cells. Although the promoter activity in FLCs was lower than that in HepG2 cells, it was higher than that in AFP-non-expressing A549 and HeLa cells, further indicating that there were AFP-expressing cells among the FLCs. Five proliferating cell colonies were generated from clonal cultures of the FLCs, and the AFP promoter activity could be detected in one of the colonies. Immunofluorescence results revealed that the percentage of AFP positive cells was (99.1±0.6)% in this clone, indicating a hepatic progenitor cells clone. Conclusion The homogenous hepatic progenitor cell clones could be selected by AFP promot