This study aimed to systematically understand the magnetic properties of magnetite (Fe₃O₄) nanoparticles functionalized with different Pluronic F-127 surfactant concentrations (Fe₃O₄@Pluronic F-127) obtained by using an improved magnetic characterization method based on three-dimensional magnetic maps generated by scanning magnetic microscopy. Additionally, these Fe₃O₄ and Fe₃O₄@Pluronic F-127 nanoparticles, as promising systems for biomedical applications, were prepared by a wet chemical reaction. The magnetization curve was obtained through these three-dimensional maps, confirming that both Fe₃O₄ and Fe₃O₄@Pluronic F-127 nanoparticles have a superparamagnetic behavior. The as-prepared samples, stored at approximately 20 °C, showed no change in the magnetization curve even months after their generation, resulting in no nanoparticles free from oxidation, as Raman measurements have confirmed. Furthermore, by applying this magnetic technique, it was possible to estimate that the nanoparticles’ magnetic core diameter was about 5 nm. Our results were confirmed by comparison with other techniques, namely as transmission electron microscopy imaging and diffraction together with Raman spectroscopy. Finally, these results, in addition to validating scanning magnetic microscopy, also highlight its potential for a detailed magnetic characterization of nanoparticles.