The invention discloses a limbus corneae stem cell serum-free medium and a culture method thereof. The serum-free medium comprises a basic culture medium and additives, wherein the additives include human recombinant EGF, insulin, 3,3',5-triiodo-L-thyronine, hydrocortisone, forskolin, manganese sulfate monohydrate, sodium selenite, sodium metasilicate, ammonium metavanadate, nickel chloride hexahydrate, stannous chloride dihydrate, ethanol amine, phosphorylethanolamine, ammonium molybdate tetrahydrate, 4-hydroxyethylpiperazine ethane sulfonic acid, vitamin C, bovine serum albumin, lipid concentrate and serum substitution. The serum-free medium contains no fetal calf serum or any animal source ingredient, can supply sufficient nutrients and good environment needed by cell growth proliferation and can effectively replace the effect of serum, cells grow well, the cell purity and stability are improved, rapid and stable cell sources are provided for limbus corneae stem cell specificity mechanism research and transplantation therapy, and the clinic application prospect is wide.