Nanoparticle-hydrogel systems have recently emerged as a class of interesting hybrid materials with immense potential for several biomedical applications. Remarkably, the incorporation of nanoparticles into a hydrogel may yield synergistic benefits lacking in a singular system. However, most synthetic strategies require laborious steps to achieve the system, severely restricting the process of translational research. Herein, a facile strategy to access a two-in-one system comprising two distinct polyurethane (PU)-based micellar systems is demonstrated and applied as a novel sustained gene delivery platform, where the two PUs are synthesized similarly but with slightly different compositions. One PU forms cationic micelles that complex with plasmid DNA (pDNA), which are loaded into a thermogel formed by another PU micellar system for the prolonged release of pDNA micelleplexes. Specifically, a thermogelling multiblock PU copolymer (denoted as EPH) was synthesized via the step-growth polymerization of poly(ethylene glycol), poly(propylene glycol), and poly(3-hydroxybutyrate). By further introducing a cationic extender, 3-(dimethylamino)-1,2-propanediol, into the reaction feed, a series of cationic PUs (denoted as EPHD) with varying compositions were obtained. The EPHDs formed positively charged micelles in aqueous solutions, efficiently condensed pDNA into nano-sized micelleplexes (<200 nm) at optimized w/w ratios, and mediated transient green fluorescence protein expression in HEK293T cells at 48 h post-transfection. On the other hand, aqueous EPH solution (4 wt %) was injectable at 4 °C and rapidly gelled upon heating to 37 °C to form a stable hydrogel depot. EPHD/pDNA micelleplexes were easily loaded into EPH by mixing the solutions at 4 °C, before heating to 37 °C, leading to the resultant hydrogel system. The in vitro release study revealed that while free pDNA loaded in the thermogel was completely released in 2 weeks, the release of EPHD/pDNA micelleplexes was prolonged to at least 28 days, suggesting substantial micelleplex-hydrogel interactions. Intact, bioactive, and noncytotoxic EPHD/pDNA micelleplexes in the release media were proved by gel retardation, in vitro gene transfection, and CCK-8 cytotoxicity assay results, respectively. Collectively, this work presents a simple approach to achieving and optimizing a novel two-in-one nanoparticle-hydrogel system for the prolonged delivery of pDNA and may be promising for long-term gene delivery applications.