The invention provides a preparation method of mouse bone marrow macrophages. The preparation method comprises the steps as follows: (1) preparing a complete medium; (2) dislocating cervical vertebraof mice of 6-9 weeks old; (3) taking femur and tibia of the mice, blowing out mouse bone marrow cells, repeating the operation until the bone is colorless, collecting a single cell suspension after blowing, performing centrifuging, and discarding a supernatant; (4) adding a red blood cell lysis buffer to cell precipitate for treatment; (5) adding the mixed solution to a complete medium rapidly forneutralization, performing centrifuging, and discarding a supernatant; (6) adding the complete medium to centrifugal cell precipitate to resuspend cells; (7) inoculating a petri dish with the resuspended cells for culture; (8) after 4 hours, sucking out the suspension cells, inoculating a petri dish of the complete medium containing an inducer with the cells, and recording the day as the first day; (9) changing half of the medium of the cells on the third day, changing the medium every other day until the seventh day, and performing testing. A simple, economical and rapid method for separating the mouse bone marrow macrophages is provided.