The invention relates to a protein-free medium suitable for large-scale cultivation and antibody production of NSO cells, which is a protein-free medium provided with clear chemical compositions. The compositions of the protein-free medium comprises 21 types of amino acid, 13 types of vitamin, 8 types of salt, 4 types of fat, 6 types of microelement, progesterone, sodium bicarbonate, Hepes, glucose, a citric acid, taurine, thymidine, adenine, hypoxanthine, phenol red, mercaptoethanol and Pluronic F-68. The protein-free medium has the advantages that the protein-free medium can support long-term subculture of the NSO cells without the necessity of adaptation, can well support normal growth and antibody expression of the NSO cells, does not contain proteins, various hydrolysate and animal origin compositions, has clear chemical compositions, is favorable for separation and purification of products, improves the quality of medical and biological products, and is convenient to prepare and suitable for mass production and application.