Objective To simplify and optimize the method for isolation and culture of primary mouse hepatocytes on the basis of conventional extraction method, and to provide a reference for related research. Methods Mouse liver was reversely perfused with the isolation solution (i.e., through the vena cava inferior in, and portal vein out), cut into small pieces and digested with enzymes. Then the hepatocytes were isolated by density gradient centrifugation and transferred into culture medium. The cell viability was detected by trypan blue staining. The purity of the hepatocytes was analyzed by flow cytometry and the cell morphology was observed with an inverted microscope. Results The hepatocytes obtained by this improved method showed high viability and purity, with typical characteristics of cell morphology. Conclusions The liver perfusion is facilitated by reversed perfusion, and the isolated hepatocytes are with high viability and purity confirmed by many times of experiments. This optimized procedure is an easy and efficient method for isolation of primary mouse hepatocytes.