In this study, a new kind of antibiotic drug-polymer conjugate, the kanamycin-methyl acrylate-polyethylenimine (KMP) polymer, was synthesized and characterized as gene carrier. Previous reports have shown that the modification of low molecular weight polyethylenimine (LMW-PEI) improves the transfection efficiency and decreases the toxicity. Kanamycin can be hydrolyzed under an acidic environment owing to the glycosidic bond. In this physicochemical study, the properties of the kanamycin-based hybrid polymers conjugated with various LMW-PEIs, such as 0.6, 1.2, and 2 kDa polyethylenimine (PEI), were investigated by 1H NMR, FT-IR, and their buffering capacity was assayed by acid-base titration method. Gel electrophoresis and picogreen assay were performed to evaluate the ability to bind with plasmid DNA. The polyplexes were nanosized with a net positive charge. The cytotoxicity and transfection efficiency were evaluated using human cervical carcinoma (HeLa) cells. KMPs exhibited lower toxicity than PEI 25 kDa, and higher transfection efficiency compared to the native LMW-PEIs.