Human ribosomal gene (hrDNA) targeting vectors ( 10～14 kb) constructed by the group are novel non-viral vectors which could specifically integrate into the ribosomal loci and characterized by their security and stable expression of therapeutic genes. However, the low transfection efficiency handicapped their clinical application. Although nuclear localization signals (NLS) could facilitate the nuclear entry of non-viral vectors and improve the transfection efficiency, the expression of therapeutic gene varied dramatically with the coupling methods and the type of chemistry used. The hrDNA vectors were conjugated by succinimidyl-[4-(psoralen-8-yloxy)]butyrate (SPB) with Simian Virus 40 NLS (SV 40 NLS) peptide through electrostatic interaction efficiently, which could protect the plasmid DNA(pDNA) from degradation of DNase. The polyethylenimine (PEI), which is an economical and low toxic polymer and wildly used in vivo gene therapy, was employed to transfect the primary human dermal fibroblasts (HDF). When conjugated with NLS peptide, the 12 kb hrDNA emerged in the nucleus within 60 min under the view of confocal microscopy. The GFP fluorescence analysis by flow cytometry showed that the transfection efficiency was increased to 4～5 folds. In conclusion, an effective procedure was developed to improve the non-viral transfection efficiency and promise the preclinical trial of hrDNA vectors.