We investigate the complexation ability of CH3I quaternized HOOC-poly(N-isopropylacrylamide)-b-poly(2-(dimethylamino) acrylate)-C12H25 (HOOC-PNIPAM-b-QPDMAEA-C12H25) thermoresponsive-cationic diblock copolymers, having different composition of the blocks, with linear DNA molecules of different lengths. The thermoresponsive-cationic diblock copolymers are able to self-assemble into nanosized aggregates in aqueous media, where the PNIPAM block possesses the inner part and the QPDMAEA block constitutes the outer part, even at ambient temperature (temperature below the LCST value of PNIPAM block) because of the presence of hydrophobic C12H25 groups placed at the QPDMAEA free chain end. Thermoresponive-cationic copolymer/DNA polyplexes were prepared at various N/P (amine over phosphate groups) ratios utilizing two DNAs of different lengths (DNAshort ≈ 113 bp and DNAlong ≈ 2000 bp). Fluorescence measurements of ethidium bromide quenching as well as UV–vis measurements reveal the complexation of the cationic polymeric aggregates with DNA molecules. In addition, dynamic and electrophoretic light scattering measurements show the structural features and the surface charge of the formed polyplexes at both 25 °C and 45 °C (temperature above the LCST value of PNIPAM block). The salt tolerance of the formed polyplexes was also examined by dynamic light measurements. The overall physiochemical characterization of the polyplexes provides new insights into the parameters affecting the interactions between thermoresponsive-cationic polymer aggregates and nucleic acids.