Novel pH- and temperature-responsive double-hydrophilic diblock copolymers, poly(ethylethylene phosphate)-block-poly[2-(dimethylamino)ethyl methacrylate] (PEEP-b-PDMAEMA), have been synthesized via the combination of ring-opening polymerization (ROP) and atom transfer radical polymerization (ATRP). The PEEP block with a bromine-terminated end (PEEP-Br) was first prepared by ROP of 2-ethoxy-2-oxo-1,3,2-dioxaphospholane (EEP) using 2-hydroxyethyl 2-bromoisobutyrate as a bifunctional initiator and stannous octoate as a catalyst. ATRP was then used to polymerize DMAEMA monomer in a methanol/water mixture with PEEP-Br as a macroinitiator, resulting in diblock copolymers of PEEP-b-PDM A EM A. Their chemical structures were respectively characterized by H-1 NMR, C-13 NMR, P-31 NMR, and FT-IR. Their molar mass distributions were determined by gel permeation chromatography (GPC). The critical aggregation concentration (cac) of PEEP-b-PDMAEMA in aqueous solution, which was measured by the fluorescence probe technique. depends on the block composition. The results measured by static laser light scattering (LLS), dynamic light scattering (DLS), and transmission electron microscopy (TEM) reveal that these diblock copolymers are able to self-assemble into aggregates with different particle sizes and morphologies in aqueous solutions, depending on various pH media. On the other hand, the UV vis measurement shows that these diblock copolymers exhibit a reproducible temperature-responsive behavior with a lower critical solution temperature (LCST) that is tunable by the block composition and pH. In addition, agarose gel retardation assays, TEM, and zeta potential measurements demonstrate that such double-hydrophilic diblock copolymers can effectively condense DNA, potentially useful for the gene delivery.