The effective biofunctionalization of nanoparticles is crucial for biomedical applications. In this study we investigated the covalent biofunctionalization of magnetic nanoparticles based on carbodiimide activation. An important aspect in the covalent biofunctionalization of nanoparticles has been neglected, namely pre-concentration. Exploiting the electrostatic attraction forces between a protein and the nanoparticle surface will favor the covalent immobilization. We showed that low ionic strength buffers with a pH slightly lower than the pI of the selected biomolecules is needed to increase the yield of covalent immobilization. Additionally, it is demonstrated that the covalently immobilized proteins are bioactive, relying on a sandwich assay using gold nanoparticles as reporter labels.