The invention discloses a CHO cell protein-free medium. The CHO cell protein-free medium comprises the following raw materials: alanine, arginine, aspartic acid, asparagine, cystine, cysteine, glutamine, glutamic acid, glycine, histidine, calcium chloride, magnesium sulfate, potassium chloride, monopotassium phosphate, disodium hydrogen phosphate, sodium bicarbonate, sodium hydroxide, ferrous sulfate heptahydrate, magnesium chloride hexahydrate, copper sulfate, sodium pyruvate, manganous chloride, sodium selenite, ammonium molybdate, nicotinamide, para aminobenzoic acid, folic acid, ribose, uracil, lipoic acid, linoleic acid L, cholesterol, dexamethasone, estradiol progesterone, testosterone and the like. The CHO cell protein-free medium does not contain serum ingredient or animal or plantsource protein, contains amino acids, vitamins, inorganic salt, organics and a small amount of hormone and facilitates purification of protein, cells do not go through the process of serum reduction,the cell activity can be maintained for a long time, and the protein expression and production time is prolonged.