The invention discloses a method for in-vitro culture of HEK293 for transient expression of protein. The method comprises the following steps: S1, resuscitating cryopreserved HEK293 cells in an original culture medium, and incubating the cryopreserved HEK293 cells in an incubator; S2, inoculating the cells into an HEK293 serum-free culture medium for culturing; S3, carrying out passage operation;S4, carrying out passage operation again; S5, preparing transfection plasmids; S6, acquiring data including cell density and survival rate, and diluting the cells; S7, diluting plasmid DNA; S8, diluting a transfection reagent; S9, adding the diluted transfection reagent into the diluted plasmid DNA, and incubating the mixture at room temperature; S10, slowly adding the mixture into the cells in the step S6, and performing shaking; and S11, carrying out culturing in an incubator, and harvesting the HEK293 cells and expression products. According to the invention, the expression quantity can beobviously increased, the expression efficiency is high, and more protein can be obtained in a shorter time.