Due to the numerous biological applications of magnetite (Fe3O4) nanoparticles (MNPs), it is essential to identify the influence of these nanoparticles on basic biological processes. Therefore, in this research, the effect of MNPs on the structure and activity of hen egg white lysozyme (HEWL) (EC 18.104.22.168) as a model protein was examined using tryptophan intrinsic fluorescence, UV/Vis, and circular dichroism spectroscopy. Moreover, enzyme activities were analyzed by a turbidometric approach in the presence of MNPs at concentrations providing MNPs/HEWL ratios in the range of 0.04–1.25. As-synthesized MNPS were characterized by Fourier transform infrared spectroscopy, x-ray diffraction, scanning electron microscopy, transmission electron microscopy, vibrating sample magnetometry and the zeta potential of MNPs was measured to be −29 mV. The goal of this work was investigating the ordering or disordering effect of MNPs on protein structure at ratios lower or higher than 0.918 as concentration ratio of threshold (CRT), respectively, in order to answer the question: 'How can the denaturation and refolding of a model protein (HEWL) be affected by MNPs?' As has been reported recently, the protein folding, helicity, and half-life were improved at <CRT to make the protein more ordered and conversely, HEWL was unfolded, and the helicity and half-life were decreased at >CRT to make the protein more disordered upon interaction with MNPs. The disordering effect of urea at >CRT and even at <CRT in the denaturation buffer (urea 6 M) increased and at <CRT the MNPs can provide a significant improvement in the refolding of the unfolded urea treated protein. These observations provide a new perspective on the growing applications of MNPs in biotechnology and biomedicine.