The investigation and clarification of the properties of surface-functionalized superparamagnetic nanoparticles in a biological environment are key challenges prior to their medical applications. In the present work, electron paramagnetic resonance spectroscopy (EPR) combined with the spin labeling technique was utilized to better understand the behavior of nitroxides attached to magnetite nanoparticles dispersed in body fluid. EPR spectra of spin-labeled, silane-coated Fe3O4 nanoparticles in human serum and whole blood were recorded and analyzed for both room- and low-temperature values. In all cases, the obtained EPR signal consisted of a broad line from magnetite cores and a characteristic signal from the attached 4-Amino-2,2,6,6-tetramethylpiperidine-1-oxyl (4-amino-TEMPO). Even for liquid samples, the anisotropic components of magnetic tensors did not fully average out, which was reflected in the differences in the intensity of three narrow hyperfine lines from nitroxide. At 230 K the irregular slow-motion signal from the attached radical was also simulated using the EasySpin toolbox, which allowed to determine the parameters related to magnetic tensors and the dynamics of the spin label. The study showed that the anisotropy of the motion of the spin label 4-amino-TEMPO reflects its interactions with the surrounding medium and the manner of the attachment of the nitroxide to the surface of nanoparticles.