The invention provides a method for preparing an immortalized duck embryo liver cell line, the method comprising: (1) digesting the duck embryo liver with a trypsin digestion method, cultivating the digestive juice, and obtaining primary cultured duck embryo liver cells; (2) Using liposome transfection method, the eukaryotic expression plasmids encoding hTERT and Marker genes were introduced into the primary cultured duck embryo liver cells; (3) The transfected duck embryo liver cells were cultured, and the G418 was used to screen the cells hTERT-positive clones; (4) culturing the screened hTERT-positive clones for more than 50 passages to obtain immortalized cell lines. The cell line still has good vigor after more than 50 passages in vitro, maintains division and proliferation, does not undergo senescence and apoptosis, and is an immortalized cell line. The immortalized duck embryo hepatocyte has regular shape, simple culture conditions, easy control and convenient use, and provides a good carrier for studying duck diseases, especially duck hepatitis virus.