Objective To prepare a single lobe of liver decellularized extracellular matrix bio-derived scaffold and to perform preliminary identification. Methods Twenty adult SD rats were randomly divided into two groups decellularization and control groups. In decellularization group, intravenous catheters were inserted through the portal vein to establish channels for a single lobe of liver perfusion successively with heparinized PBS, 1%Triton X-100（pH 7.5-8.0）and PBS in 37℃. After decellularization, the scaffold and native liver were observed through genomic DNA contact analysis, scanning electron microscope, HE and Masson’s staining. Immunofluorescence staining and vascular cast were used to observe changes in extracellular matrix constitution. Results The single lobe of liver was decellularized in 4 hours. Its cells and debris were cleaned gradually in the perfusion process and became translucent eventually. HE, Masson’s, immunohistochemistry staining and scanning electron microscope showed a lot of collagen fibers but no visible cell nuclei remained after decellularization. Quantitative analysis of DNA content within the scaffold showed a 97.32% decrease compared to the native liver. The agarose gel electrophoresis showed no DNA bands associated with the decellularized scaffold. Cast specimen showed that portal vein was still intact and clear compared with the native liver. Conclusion The method of perfusion with Triton X-100 can effectively remove all cellular components, and retain the extracellular matrix and vascular network structure well. It is a convenient and ideal preparation method to decellularize a single lobe of liver scaffold with tissue engineering.