Here, the efficiency of magnetic nanosphere (Fe3O4) coated with L-histidine (L-his) was evaluated to enrich proteins of low-abundance in human serum. The chemometric analysis provided optimal conditions to concentrate low-abundance proteins, reducing the dynamic range of protein levels in the serum sample. Three independent factors (temperature, mass ratio and pH) were tested. The optimal condition achieved was using temperature (42 °C), ratio (3:1, nanomaterial/sample) and pH (5.5). The kinetic study revealed that the adsorption of proteins on the surface of the monolayer magnetic nanosphere follows a pseudo-second order model with R2 = 0.9153. Adsorption isotherm experiments revealed high capacity of protein adsorption on the surface of the nanosphere, indicating a maximum theoretical adsorption capacity (qmax) of 123.45 mg g−1. The efficiency of enrichment of low-abundance proteins in the serum sample was evaluated by gel electrophoresis (SDS-PAGE). Furthermore, when compared to the method using organic solvent (acetonitrile), Fe3O4@L-his showed adequate efficiency to simplify the serum sample. Using MALDI-TOF/TOF, approximately 50 proteins were identified using Fe3O4@L-his. As a proof of concept for this study, patients with bipolar disorder and healthy controls were discriminated using the magnetic nanosphere as an adsorbent in the sample preparation step. These results show that Fe3O4@L-his can be a fast, robust, and simple alternative to enrich low-abundance proteins in human serum, emerging as an important strategy in the search for biomarkers of human diseases.