The invention discloses a culturing medium for cultivating animal cells without serum and with low density and an application thereof. The culturing medium for cultivating animal cells without serum and with low density is characterized in that synthetic culturing medium is adopted as the raw material, and insulin powder, transferrin powder, L-glutamine powder, sodium selenite powder, ethanolamine solution, lipid solution, epidermal growth factor powder, a recombinant human acidic fibroblast growth factor, vitamin solution, plant hydrolysate solution, putrescine, ferrous sulfate, hydrocortisone and an insulin-like factor are added into the synthetic culturing medium. The culturing medium can be suitable for the growth and metabolism and product expression of the cells of the mammals in the condition of low density without the serum. Combined with the method that D- polylysine powder is coated on a culture plate with 96 holes, the use of the culturing medium for cultivating animal cells without serum and with low density can clone the animal cells without the serum, thereby realizing the continuity of cultivating the animal cells without the serum, improving the stability of product expression, assuring the quality and safety of the product, and enhancing the controllability of the production process. Thus, the culturing medium for cultivating animal cells without serum and with low density has wide application range.