Efficient and safe gene vectors are important for gene therapy. Here, a novel family of amphiphilic polyethylenimine (PEI) LD1-PEI bearing a polar group of branched PEI 25K and four dodecyl chains was developed. Agarose gel electrophoresis was used to confirm the formation of complexes. The transfection activity of the amphiphilic carrier was evaluated in different cell lines. The in vitro study showed that LD1-PEI showed a higher transfection efficiency with improved biocompatibility than PEI 25K. Serum showed almost no or only a slight effect on LD1-PEI/DNA transfection efficiency. In summary, LD1-PEI is a promising nonviral gene carrier.