The invention discloses a culture solution for culturing 293T cells in a serum-free suspension mode. According to the culture solution for culturing the 293T cells in the serum-free suspension mode, ahigh-glucose DEMEM-F12 culture substrate without L-glutamine serves as a base solution, and L-alanyl-L-glutamine, vitamin C, HEPES, an insulin-like growth factor, an epidermal growth factor, lipid concentrate, human serum albumin, transferrin, trehalose, heparin sodium and Pluronic F-68 are added. The culture solution for culturing the 293T cells in the serum-free suspension mode contains clear components without any serum components, is explicit in component, stable in batch, controllable in quality, low in cost and suitable for not only routine culture in laboratories but also large-scale production culture, and has broad application prospects.