This work presents synthesis and characterization of sophorolipid-coated monodisperse iron oxide nanoparticles. Sophorolipids are biological glycosylated amphiphiles produced by the yeast S. bombicola. In their open acidic form, sophorolipids have been used as surface stabilizing agent for metal and metal oxide nanoparticles but with poor control over size and structural properties. In this work, the COOH function of sophorolipids (SL) was modified with nitrodopamine (NDA), a catechol known for its high affinity to iron ions. The resulting new form of sophorolipid-nitrodopamide (SL-NDA) was used as surface ligand for monodisperse iron oxide nanoparticles. We show, by a combination of thermogravimetric analysis and small angle X-ray and neutron scattering, that iron oxide nanoparticles (IONP) are stabilized by a single, high-density SL-NDA layer, which results in excellent colloidal stability under biologically relevant conditions such as high protein and salt concentration. The IONP grafted with SL-NDA showed negligible uptake and no cytotoxicity tested on two representative cell lines. Thus, they reveal the potential of sophorolipids as stable and non-toxic surface coatings for IONP-based biomedical and biotechnological applications.