A new multifunctional gene delivery system was constructed with diphtheria toxin’s functional domains. Used functional domains are T domain for endosomal escape and R domain for efficient internalization into cell. In order to conjugate these domains into PEI polyplex, diphtheria toxin T and R domains-streptavidin fusion protein (DTRS) was prepared. The conjugation of the DTRS with biotinylated PEI polyplex (DTRS-polyplex) lead to the significant enhancement of transfection efficiency when compared with plain PEI/pDNA polyplex in CHO-K1 cell. It was demonstrated that DTRS-polyplex had high endosomal escape efficiency and internalization efficiency by several measurements, such as in vitro intracellular trafficking observation and the internalization inhibition with several inhibitors. These results suggest that this multifunctional non-viral vector may contribute to the future cancer gene therapy.