The invention provides polyethyleneimine-polylysine copolymer which has the structure of a formula (I). The invention also provides a preparation method of the polyethyleneimine-polylysine copolymer. The method comprises the following steps of: carrying out polymerization reaction to the polyethyleneimine and epsilon-carbobenzoxy-L-lysineN-carboxylic acid anhydride in a first organic solvent so as to obtain polyethyleneimine poly-epsilon-carbobenzoxy-L-lysine copolymer; and mixing the halogen hydride solution, a second organic solvent and the polyethyleneimine poly-epsilon-carbobenzoxy-L-lysine copolymer, and reacting so as to obtain the polyethyleneimine-polylysine copolymer with the structure of the formula (I). The polyethyleneimine-polylysine copolymer provided by the invention is a novel polycation gene carrier, the properties of the polyethyleneimine and the polyamino acid are integrated, and thus the copolymer has relatively high transfection efficiency.