The present study is focused on the synthesis and characterization of nitric oxide (NO)-releasing superparamagnetic iron oxide nanoparticles (Fe₃O₄ NPs), and their incorporation in Pluronic F127 hydrogel with great potential for topical applications. Magnetite nanoparticles (Fe₃O₄ NPs) were synthesized by thermal decomposition of acetylacetonate iron (Fe(acac)₃), and coated with the thiol containing molecule mercaptosuccinic acid (MSA), leading to Fe₃O₄-MSA NPs. The obtained NPs were characterized using different techniques. The results showed that the Fe₃O₄-MSA NPs have a mean diameter of 11 nm, in the solid state, and superparamagnetic behavior at room temperature. Fe₃O₄-MSA NPs have an average hydrodynamic size of (78.0 ± 0.9) nm, average size distribution (PDI) of 0.302 ± 0.04, and zeta potential of (−22.10 ± 0.55) mV. Free thiol groups on the Fe₃O₄-MSA NP surface were nitrosated by the addition of sodium nitrite, yielding S-nitrosated magnetic nanoparticles (Fe₃O₄-S-nitroso-MSA NPs), which act as spontaneous NO donors upon S–N bond cleavage. The amount of (86.4 ± 4.7) μmol of NO was released per gram of Fe₃O₄-S-nitroso-MSA NPs. In order to enhance NP dispersion, Fe₃O₄-MSA NPs were incorporated in Pluronic F127 hydrogel (3.4% w/w), and characterized using different techniques. Rheological measurements suggest a potential use for Fe₃O₄-MSA NPs dispersed in Pluronic hydrogel for topical applications. Atomic force microscopy (AFM) showed that the NPs are embedded within the Pluronic film while the X-ray photoelectron spectroscopy (XPS) spectrum of the Fe₃O₄-MSA NPs samples revealed the presence of iron, oxygen, carbon and sulfur, confirming the presence of MSA molecules on the NP surface.