The invention discloses a serum-free CHO cell culture medium and a preparation method thereof. By adding such supplement factors with determined chemical ingredients as a variety of amino acids, vitamins, inorganic salts, trace elements, alkaloids, fatty acids and hormone and the like, the culture medium can be used for quickly growing CHO cells under a suspension culture condition without adaptation, the cell density and cell activity can be equal to those of a cell culture with serum, the CHO cell culture requirements can be satisfied and multiple unfavorable factors caused by the use of serum can be effectively avoided, the production cost is reduced and the serum-free CHO cell culture medium can be completely replacing the cell culture with serum to perform large scale culture and production of CHO cells. In addition, due to a powdery storage manner, the product transport risk can be effectively avoided, the transport cost can be reduced and the requirements of performing large scale culture on the CHO cells can be satisfied.