Abstract: Objective: To compare and define the suitable range of the different Methods for isolation of murine hepatic lymphocyte，natural killer T cells( NKT cells) and natural killer cells( NK cells) . Methods: According to factorial design， four liver pretreatmentMethods including unbleeding method( A method) ， the eyeballs bleeding method( B method) ，body perfusion method( C method) ， the eyeball with body perfusion method( B method + C method) and two percoll isolation Methods including single concentration method( Ⅰ method) and continuous density gradient method( Ⅱ method) had been combined into 8 kinds of combination conditions， four bleeding Methods were used to prepare cell suspension respectively，and the single concentration method ( 33% Percoll) and the continuous density gradient method( 33% Percoll + 70% Percoll) were used to separation of mouse hepatic lymphocytes respectively，the hepatic innate immune cells were analyzed with two or three-colors immunofluorescent-labeling. Results: With the analysis of variance of factorial design， the highest proportion of lymphocytes was obtained by B method + C method， the highest absolute lymphocyte count was obtained by A method， the highest NKT and NK cell percentage and absolute number was obtained through B method; the ratios and the number of lymphocyte，T cell，NKT cell，NK cell obtained from B method were higher than those of A method. The ratio of NKT cell and NK cell absolute count， liver pretreatment method and percoll concentration had interactions each other( P ＜0. 01 and P＜0. 05)， the others were no interaction( P ＞0. 05) . Conclusions: High proportion of natural immune cells can be obtained by bleeding with perfusion combined with the continuous density gradient method( 33% + 70%)， it is helpful for research on NKT cells and NK cells.