The invention provides a separation and culture method of renal podocyte of a mouse. The separation and culture method comprises the following steps of (a), intraperitoneally injecting 2% pentobarbital sodium to execute the mouse, sterilizing the mouse with alcohol, taking out kidney tissue of the mouse, putting the kidney tissue into a precooled sterile PBS (Phosphate Buffer solution) containing double antibodies, soaking and washing the kidney tissue, and peeling and removing a renal capsule; (b), mechanically dissociating the kidney tissue, then digesting the kidney tissue by a preheated mixed enzyme solution, and terminating digestion by a mixed complete culture solution; (c), obtaining the renal podocyte by adopting a difference sieving method, inoculating the podocyte in a treated cell bottle, and culturing the podocyte; (d), digesting a cell, which is cultured for 3d, through pancreatin, then filtering the cell once again by a sieve mesh, centrifuging filtrate to remove supernatant fluid, and resuspending and inoculating an obtained mixture in the treated cell culture flask; (e), identifying the renal podocyte by cell immunofluorescence. The separation and culture method of the renal podocyte of the mouse, which is provided by the invention, is good in repeatability, and is simple and convenient to operate; moreover, the high-yield and high-purity renal podocyte of the mouse can be obtained.