The invention discloses a culture method of AD293 spherical cell cluster. The culture method comprises the following steps: (1) preparing a suspension culture medium: preparing the following components: a serum-free culture medium, fetal calf serum, a serum replacement, a non-essential amino acid solution, epidermal growth factor, transforming growth factor, and methyl cellulose, and evenly mixing and dissolving the components mentioned above to obtain a suspension culture medium; (2) preparing AD293 cells: using a DMEM culture medium with 10% of fetal calf serum to revive AD293 cells in a cell culture flask, and carrying out cell passage; (3) culturing the cells: adding AD293 cells obtained in the step (2) and suspension culture medium obtained in the step (1) into a shake flask, then placing the shake flask on a shaking bed, and culturing the cells for 48 to 72 hours at a temperature of 37 DEG C to obtain AD293 spherical cell clusters. Compared with the cells cultured by spherical suspension micro carriers, the cell density of obtained AD293 cell clusters is increased by 10 times or more.