A separation culture method for lens epithelium stem cells is disclosed. The method includes following steps: (1) cutting a lens anterior capsule into small pieces, digesting, and paving the obtained cells to a culture flask coated with a medium, a culture plate coated with a medium or a culture dish coated with a medium; and (2) adding an MEM culture solution containing 15-25% of FBS, 50-150 mug/L of a human epidermal growth factor (FGF), 5-10 mg/L of insulin, 5-10 mg/L of hydrocortisone, 5-10 mg/L of a vibrio cholera toxin, 0.01-0.05 mg/L of 3,3',5-iodo-L-para-hydroxyphenylalanine, 1-5 mg/L of adenine, 5-10 mg/L of glycine, 6-12 mg/L of alanine, 10-16 mg/L of asparagines, 10-16 mg/L of aspartic acid, 12-18 mg/L of glutamic acid, 8-15 mg/L of proline and 7-14 mg/L of serine, putting into an incubator having a temperature of 37 DEG C, culturing for 2-3 days to form clone, and forming lens stem cells with monolayer activity after 10-14 days. A separation culture solution for the lens epithelium stem cells is also disclosed. The method can separate and obtain the primary lens epithelium stem cells. The stem cells are high in activity. The method is simple, convenient, easy in operation and good in application prospect.