【】Objective It is to explore the extraction and purification methods of Kupffer cells and hepatic stellate cells from mouse liver, and provide reference and suggestions for the separation and extraction methodology of primary non parenchymal cells from mouse liver.Methods Based on in vivo collagenase perfusion digestion, different reagents and methods such as Percoll and OptiPrep were used to extract C57BL/6 mice Kupffer cells and hepatic stellate cells and evaluate the purity through flow cytometry and immunofluorescence methods. Results The two-layer Percoll method for extracting Kupffer cells and the two-layer OptiPrep method for extracting hepatic stellate cells are feasible,and the purity can reach more than 90%.The cell yield was 1~2×107/liver, the cell survival rate was more than 90%.After 48 hours of primary cell culture, the number of Kupffer cells F4/80-positive cells and hepatic stellate cells α-SMA-positive cells reached more than 75%. Conclusions The separation and extraction method of Kupffer cells and hepatic stellate cells from mouse liver is perfect, reliable, cost-effective and reproducible.