The invention discloses a reprogramming culture medium, a preparation method thereof and a culture method of a reprogramming cell, wherein the reprogramming culture medium comprises a basal culture medium, a reprogramming additive 1 and a reprogramming additive 2, wherein the basal culture medium is a DMEM (Dulbecco Modified Eagle Medium)/F12 culture medium; the reprogramming additive 1 comprises sodium hydrogen carbonate, sodium selenate, recombinant human insulin, recombinant human basic fibroblast growth factors, recombinant human transferrin, ascorbic acid and hydrocortisone; the reprogramming additive 2 comprises sodium hydrogen carbonate, sodium selenate, recombinant human insulin, recombinant human basic fibroblast growth factors, recombinant human transferrin, ascorbic acid, valproic acid and sodium butyrate. The reprogramming culture medium disclosed by the invention is exact in component, stable in quality, high in reprogramming efficiency, wide in cell lineage and capable of reprogramming and culturing cells such as human skin fibroblasts, peripheral blood mononuclear cells, amniotic epithelial cells and the like.