Barley plants were grown in water solutions containing nutrient elements and superparamagnetic iron oxide nanoparticles (SPIONs), particularly the magnetite nanoparticles (Fe3O4; 15–20-nm particle size). No visible detection of toxicity on plants’ growth had been observed. The different samples from two different organs of the plants, i.e., roots and leaves, were investigated using X-ray diffraction (XRD), bright-field microscope, and transmission electron microscope (TEM). To track the SPIONs, magnetic measurements using vibrating sample magnetometry (VSM) and Mössbauer spectroscopy at room temperature were also performed in the two different dried root and leaf organs of the plants. The magnetic signals measured in root and leaf organs clearly indicated an uptake of Fe3O4 magnetic nanoparticles (MNPs) by the barley plant from aqueous medium containing Fe3O4 MNPs and their subsequent accumulation in the plant organs. The different parameters such as hyperfine magnetic field, quadrupole splitting, isomer shift, and line width were deduced from Mössbauer spectroscopy data. The Mössbauer spectra are constituted of two magnetic sextets (A site, B site) and paramagnetic central doublet.