Multifunctional nanoparticles with high gene transfection activity, low cytotoxicity, photoacoustic imaging ability, and photothermal therapeutic properties were prepared by conjugating low-molecular-weight polyethylenimine onto the surfaces of gold nanorods through the formation of stable S–Au bonded conjugates. Results revealed that the gene transfection efficiency of the prepared polyethylenimine-modified gold nanorods (GNRs-PEI1.8k) was higher and their cytotoxicity was less than those of the commercial reagent PEI25k. GNRs-PEI1.8k could also be potentially used as a photoacoustic and photothermal reagent to evaluate the pharmacokinetics, biodistribution, and antitumor effects of gene/drug nanoparticles. Therefore, GNRs-PEI1.8k can be considered a promising candidate for the clinical diagnosis and treatment of tumors.