The encouraging results of preliminary toxicological studies on imidazolium-based ionic liquids provide good opportunities for the development of ionic liquids in biomedical applications. In this work, the polymerized ionic liquid poly[3-butyl-1-vinylimidazolium L-proline salt] has been synthesized as a gene vector. The interaction of poly[3-butyl-1-vinylimidazolium L-proline salt] with DNA was studied by agarose gel electrophoresis. The cell viability was determined through PI (propidium iodine) staining and flow cytometry, showing marginal toxicity toward the cells examined. The transfection efficiency was evaluated through the in vitro transfection experiment. The results indicated that the imidazolium cation had a high binding ability to DNA, and the condensed DNA in the complexes could be effectively protected against enzymatic degradation. Poly[3-butyl-1-vinylimidazolium L-proline salt] could further transfer the reporter gene into the HeLa cell and successfully mediate the gene expression without the aid of additional agent.