Two series of poly(vinyl amine) (PVAm)-based block copolymers with zwitterionic and thermoresponsive segments were synthesized by the reversible addition-fragmentation chain transfer polymerization. A mixture of the two copolymers, poly(N-acryloyl-l-lysine) (PALysOH) and poly(N-isopropylacrylamide) (PNIPAM), which have the same cationic PVAm chain but different shell-forming segments, were used to prepare mixed polyplex micelles with DNA. Both PVAm-b-PALysOH and PVAm-b-PNIPAM showed low cytotoxicity, with characteristic assembled structures and stimuli-responsive properties. The cationic PVAm segment in both block copolymers showed site-specific interactions with DNA, which were evaluated by dynamic light scattering, zeta potential, circular dichroism, agarose gel electrophoresis, atomic force microscopy, and transmission electron microscopy measurements. The PVAm-b-PNIPAM/DNA polyplexes showed the characteristic temperature-induced formation of assembled structures in which the polyplex size, surface charge, chiroptical property of DNA, and polymer-DNA binding were governed by the nitrogen/phosphate (N/P) ratio. The DNA binding strength and colloidal stability of the PVAm-b-PALysOH/DNA polyplexes could be tuned by introducing an appropriate amount of zwitterionic PALysOH functionality, while maintaining the polyplex size, surface charge, and chiroptical property, regardless of the N/P ratio. The mixed polyplex micelles showed temperature-induced stability originating from the hydrophobic (dehydrated) PNIPAM chains upon heating, and remarkable stability under salty conditions owing to the presence of the zwitterionic PALysOH chain on the polyplex surface.