Background/Objectives: The study describes the elaboration and evaluation of thermosensitive niosomes intended for the systemic application of daunorubicin hydrochloride. The attained stimulus sensitivity would determine the release of the chemotherapeutic predominantly at the target site, which ensures a higher drug concentration and leads to reduced systemic toxicity. The latter is highly beneficial, as the anthracycline antibiotic is known for its dose-dependent cardiotoxic effects. Methods: Conventional and copolymer-modified niosomes were prepared via thin-film hydration and the transmembrane ammonium gradient method, allowing us to assess the impacts of copolymer type-DHP-PiPOX (1,3-dihexadecyl-propane-2-ol-poly(2-isopropyl-2-oxazoline)) or DHP-PETEGA (1,3-dihexadecyl-propane-2-ol-poly(ethoxytriethylene glycol acrylate)) and their concentrations (0.5, 1, and 2.5 mol%), as well as the method of preparation, on the main physicochemical properties of the vesicles. Niosomes were characterized in terms of their size, polydispersity index (PDI), zeta potential, entrapment efficiency, morphology, and drug release properties. Thermosensitivity was evaluated by fluorescence studies, and the antiproliferative activity of optimized formulations was assessed against the acute myelocyte leukemia-derived HL-60 cell line. Results: Daunorubicin-loaded niosomes modified with DHP-PiPOX and DHP-PETEGA at 2.5 mol% exhibited suitable physicochemical properties for systemic application, with sizes below 200 nm (155 and 158 nm respectively), low PDI values of 0.25 and 0.29, spherical morphology, and high daunorubicin entrapment efficiency (68.6 and 66.5% respectively). The vesicles showed temperature-dependent drug release properties and superior antiproliferative activity compared to the free daunorubicin (IC₅₀ values of 6.91 and 8.54 vs. 12.14). Conclusions: The obtained results indicate that the developed thermosensitive nanovesicles may serve as a suitable drug delivery system for the systemic application of daunorubicin hydrochloride.