Magnetic metal oxide nanoparticles demonstrate great applicability in several fields such as biotechnology, medicine and catalysis. A stable, magnetic and low-cost material, nanoscale magnetite, is an interesting adsorbent for protein purification. Downstream processing can account for up to 80% of the total production costs in biotechnological production. As such, the development of new innovative separation methods can be regarded as highly profitable. While short peptide sequences can be used as specific affinity tags for functionalised adsorber surfaces, they need expensive affinity ligands on the particle surface for adsorption. In order to identify peptide tags for several non-functionalised inorganic surfaces, different binding conditions to iron oxide nanoparticles are evaluated. Therefore, magnetite nanoparticles in a range of 5–20 nm were synthesised with a co-precipitation method. Zeta potential measurements indicated an amphiphilic surface with an isoelectric point in the neutral pH region. Glutamic acid-based homo-peptides were used as affinity peptides for the magnetite nanoparticles. We demonstrate a dependence of the binding affinity of the peptides on pH and buffer ions in two different experimental set-ups. The nature of surface coordination for glutamic acid-based peptides can be demonstrated with different spectroscopic approaches such as infrared spectroscopy (IR), Raman spectroscopy and circular dichroism spectroscopy (CD). We want to emphasise the importance of physicochemical properties such as surface energy, polarity, morphology and charge. These parameters, which are dependent on the environmental conditions, play a crucial role in peptide interactions with iron oxide surfaces. The understanding of the adsorption of simple biomolecules on nanoscale metal oxide surfaces also represents the key to the even more complex interactions of proteins at the bio-nano interface. From the identification of interaction patterns and an understanding of the adsorption of these peptides, the up-scaling to tagged model proteins facilitates the possibility of an industrial magnetic separation process and might therefore reduce time and costs in purification processes.