The invention discloses a mouse primary hepatocyte perfusion type separating and in-vitro culturing method. The perfusion type separating of mouse primary hepatocytes is conduced through an in-situ living body perfusion method, a high-yield single-cell suspension can be obtained, and therefore a basis is provided for in-vitro long-time culturing of the primary hepatocytes. An applied stimulating culture solution contains mouse colony stimulating factors (CSF), mouse IL-2, mouse IL-6 and mouse recombinant hepatocyte growth factors r-mHGF and other cell factors; the primary hepatocytes can be separated through stimulation according to the reasonable ratio for in-vitro proliferation, and the number can reach 2.25 times the number of initially-added primary hepatocytes 72 hours later. The cellproliferation activity can be kept for at least 1-2 weeks under the in-vitro conditions, the problem that the primary hepatocytes cannot be cultured for a long time in vitro is solved, and thereforethe method can be used for infection of retroviruses, provides a basis for over-expression target genes or knockout target genes in in-vitro primary cells, and greatly expands the application field ofthe primary hepatocytes.