Stimuli-responsiveness is an important characteristic of nanocarriers that can deliver therapeutic molecules in a controlled and on-demand manner. In particular, temperature-responsive micelles have recently gained increasing attention for use in diverse therapeutic areas, including hyperthermia therapy. Herein, we developed novel shell cross-linked Pluronic F127 (PF127) polymers with branched polyethylenimine (PEI), called FPEI, for temperature-triggered drug delivery and gene transfection. The successful synthesis of FPEI copolymers was confirmed by Fourier-transform infrared spectroscopy, with the weight percent of PEI in the FPEI copolymers being 7.5%. The critical micelle concentration of FPEI and PF127 copolymers was shown to be 0.013 and 0.03 mg ml⁻¹ at room temperature, respectively. When Nile Red (NR), used as a hydrophobic model drug, was loaded into FPEI copolymers, the appropriate concentration of NR was 3 and 5 μg ml⁻¹ in 5 and 10 mg ml⁻¹ of FPEI, respectively. Interestingly, the loading capacity of FPEI increased with an increase in the micelle concentration. With increasing temperature, the NR release from the micelle increased, while the micelle radii of FPEI, NR-loaded FPEI, and NR-loaded FPEI–DNA decreased. The DNA binding ability of FPEI copolymers increased with an increase in the N/P ratio. FPEI cytotoxicity in HeLa cells showed a similar tendency, when equivalent PEI amount in FPEI was treated. While further in-depth studies remain as to the in vitro and in vivo biological functions, the FPEI system demonstrated well temperature-triggered NR release into HeLa cells, along with sufficient transfection of plasmid green fluorescence protein, which suggested potential usefulness in temperature-responsive therapy.