Objective:To develop a convenient,inexpensive and efficiency method for separate high-purity primary hepatocytes isolation of murine. Methods:The improved method was used with low concentration type IV collagenase. Purified hepatocytes were separated from the dissociative cells by low-speed centrifugation(300 r/min,1min,3-5 times). The survival rate was measured by typan blue exclusion and the purity was measured by PAS staining. The time,viability and purity of two methods were compared. Results:The cell viability was higher than that of cells obtained by traditional method,and the quantity of collagenase and the time used was decreased markedly. The purity was nearly the same as the method of percoll purity. Conclusion:A new method for separate high-purity primary hepatocytes isolation was sullessfully established.