Abstract: The preparation of drug and gene co-delivery nanoparticles was the key scientific problem in tumor multi-combined therapy. In this research, the hydrophobic fluorescent dye rhodamine B was chosen as a model drug. PEI-g-cholesterol amphiphilic polymer was synthesized as drug carrier. By selecting proper conditions, rhodamine B and gene co-delivery nanoparticles were successfully constructed via supramolecular assembly. The co-delivery particles with diameter about 150 nm and ξ-potential about 33 mV could effectively condense DNA. The in-vitro cell culture experiments indicated that the positive co-delivery nanoparticles was easily uptaked by HEK293 cells and showed effective transfection efficiency.