The invention belongs to the technical field of cell culture, and particularly relates to a knockout serum replacement for cell culture and a preparation method thereof, a knockout serum replacement composition for cell culture and a cell culture medium. The knockout serum replacement for cell culture comprises, by weight, water, 18.5-67 parts of amino acid, 200-815 parts of protein, 62.5-252 parts of inorganic salt, 1.0-3.8 parts of ethanol amine, 50-200.0 parts of sodium pyruvate, 1.25-5.0 parts of ascorbyl phosphate, 0.5-2.0 parts of glutathione and 5.0-20 parts of insulin. The knockout serum replacement for cell culture can be added into any one cell culture medium, the adding amount of serum is reduced, and therefore, the cost is saved. The knockout serum replacement for cell cultureis suitable for protein recombination, vaccine production and other kinds of cell culture, and high in universality.