For the design of new polymeric-based drug delivery systems, understanding how multiple functionalities in the polymer structure are influencing each other in particle formation is important. Therefore in this study, the balance between hydrophobic and electrostatic interactions has been investigated for thermosensitive plasmid DNA (pDNA)-loaded polyplexes. NPD triblock copolymers consisting of a thermosensitive poly(N-isopropylacrylamide) (PNIPAM, N), a hydrophilic poly(ethylene glycol) (PEG, P) and a cationic poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA, D) block with different block lengths were prepared using a hetero-functional PEG macroinitiator. Cloud points of the thermosensitive polymers in HBS buffer (20 mM HEPES, 150 mM NaCl, pH 7.4) were determined by light scattering and ranged between 33 °C and 34 °C for the different polymers. The binding and condensation properties of these thermosensitive polymers and pDNA were studied taking non-thermosensitive PD polymers as controls. The size, surface charge, and stability of the formed colloidal particles (‘polyplexes’) were studied as a function of polymer block lengths, N/P charge ratio, and temperature. The NPD polymers were able to self-assemble into polyplex nanostructures with hydrodynamic sizes ranging between 150 and 205 nm at room temperature in HBS buffer as determined by dynamic light scattering. Polyplexes prepared with a low N/P charge ratio of 1 aggregated upon heating to 37 °C, which was not observed at higher N/P charge ratios. When the length of the cationic D block was relatively long compared to the thermosensitive N block, stable polyplexes were formed at all N/P ratios and elevated temperatures. 1H-NMR studies, static light scattering and ζ-potential measurements further supported the stability of these polyplexes at 37 °C. Finally, the presence of thermosensitive blocks in NPD-based polyplexes resulted in better cytocompatibility compared to PD-based polyplexes with similar efficiencies of delivering its cargo into HeLa cells.