Objective: To study the effects of the adding different concentrations of various components in the basal culture medium I/F/D on the in vitro activity of rhEPO-Fc.Methods: The optimal components suitable for the expression of rhEPO-Fc were screened by the single factor exptl. results combined with Box-Behnken response surface method.Results: When 1% F68, 10% vitamin, 7.7 g/L putrescine, 10.1 g/L ethanolamine and 5.38 g/L insulin were added in the basal culture medium, the in vitro activity rhEPO-Fc was increased by 108.4% up of 10 268 IU/mL compared with the results in the initial basal serum free medium.Conclusion: The in vitro activity of rhEPO-Fc can be increased when cells were incubated in the optimized culture medium, the costs for the production of rhEPO-Fc can be obviously reduced.The results of this study can provide a significant reference for pilot production of rhEPO-Fc.