One of the largest challenges in gene therapy is to develop an efficient delivery vector without cytotoxicity. In this study, we propose a new concept in exploring low-positively charged thermosensitive copolymers as gene delivery vectors. Two types of copolymers, random copolymers (RCs) and blocky random copolymers (BRCs) of P(NIPAAm-co-HEMA-co-DMAEMA), are synthesized by free radical polymerization. Dynamic light scattering (DLS) and light transmittance measurements demonstrate the thermosensitive property and sharp phase transition behavior of the synthesized copolymers. Both RCs and BRCs exhibit proton buffering capacity. Cell viability assays indicate the low cytotoxicity from BRCs and almost no cytotoxicity from RCs which is ascribed to the presence of low positive charges along copolymer backbones. Such properties facilitate plasmid DNA (pDNA) delivery applications. The complexation of pDNA and RCs is detected by agarose gel electrophoresis and DLS. Furthermore, RCs are used as DNA delivery vectors to deliver green fluorescent protein (GFP) gene into HEK293T cells. The transfection efficiency is evaluated by GFP protein expression, which is affected by the charge content and transfection operation temperature. Lowering the temperature to 25 °C during the transfection course improves transfection efficiency, which results from the polyplexes dissociation at the temperature below the lower critical solution temperature (LCST). Our results reinforce that the new concept for efficient gene delivery without cytotoxicity at the transfection dose is achieved by using low-positively charged thermosensitive copolymer vectors.