The invention discloses a HeLa-F cell, and the cell is characterized in that the cell is a human cervical carcinoma cell HeLa-F whose preservation number in China Center for Type Culture Collection is CCTCC C2015138. The invention also discloses a recombinant HeLa-F cell and a method for preparing vaccinia virus by using the HeLa-F cell. The HeLa-F cell is a suspension cell, high density large scale suspension culture can be directly carried out for the cell; and the same time the cell has good capability for supporting replication of virus, and can be used for preparing a large amount of virus products and high quality vaccine products with low cost.