Objective To explore the extraction and purification method of Kupffer and hepatic stellate cells from mouse liver and provide references and suggestions for the separation and extraction method ology of primary nonparenchymal cells from mouse liver. Methods After in vivo collagenase perfusion digestion, various reagents and method, such as Percoll and OptiPrep, were used to extract C57BL/6 mice Kupffer and hepatic stellate cells, and evaluate their purity by flow cytometry and immunofluorescence. Results The two-layer Percoll method to extract Kupffer cells and the two-layer OptiPrep method to extract hepatic stellate cells were feasible, and purity reached >90%. The cell yield was 1~2×10⁷ / liver, and the cell survival rate was >90%. After 48 hours of primary cell culture, the number of F4/80-positive Kupffer cells and α-SMA-positive hepatic stellate cells reached > 90%. Conclusions The separation and extraction method of Kupffer and hepatic stellate cells from mouse liver are perfect, reliable, cost-effective, and reproducible.