The invention discloses a culture medium for hepatoma organ cell sphere culture. The culture medium is composed of a basic culture medium, 10-25 ng/mL of an epidermal growth factor, 10-25 ng/mL of a fibroblast growth factor, 0.5-2 ng/mL of a cell growth factor, 1-3 ng/mL of an insulin growth factor, 1 mmol/L of sodium pyruvate, 2 mmol/L of glutamine, 100 U/mL of penicillin, 100 [mu]g/mL of streptomycin and 10% by volume of serum. Aiming at the culture growth characteristics of liver cancer tissue-derived cells, the culture medium provided by the invention is simple and reasonable in components, rich in nutrition and moderate in formula price; and hepatoma organ cell spheres cultured by the culture medium can effectively and stably grow for a long time, are closely gathered, have smooth andround surfaces and clear boundaries, is free of disintegration phenomenon, and can be significantly improved in cell activity, specific functions and metabolic functions.