The invention provides a separation, purification and culture method of rat cornea endothelial cells, and belongs to the field of cell biology. The method comprises the following steps: 1, separating a cornea tissue sample to obtain single cells; 2, preparing immunomagnetic beads for specifically adsorbing cornea endothelial cells, and purifying the cornea endothelial cells from the separated single cells; and 3, culturing the rat cornea endothelial cells with an optimized cell culture liquid. The separation process of the rat cornea endothelial cells has the advantages of simplicity in operation, high cell output and high cell survival rate, the purification process of the rat cornea endothelial cells has the advantages of simplicity in operation, very high yield and very high purification rate, and the culture process of the rat cornea endothelial cells has the advantages of fast cell proliferation, short period and high economic benefit.