In this work, novel Fe^III–tannic acid (TA) film-functionalized magnetic silica microspheres (designated Fe₃O₄@SiO₂@FTA) were prepared via a facile deposition of one-step assembled Fe^III–TA films on the surface of magnetic silica microspheres. The resulting Fe₃O₄@SiO₂@FTA microspheres possessed strong magnetic responsiveness and good dispersity in aqueous solutions. We introduced low concentrations of standard peptide, standard protein, peptides in BSA tryptic digests and human urine to investigate the enrichment capability of the prepared nanocomposites. The enrichment process was very fast (only 5 min) and the peptide/protein–magnetic microsphere conjugates were readily separated by an external magnet. Moreover, the obtained conjugates can be directly analyzed by MALDI-TOF MS without any elution procedure. The enrichment performance was quite satisfying, indicating Fe₃O₄@SiO₂@FTA microspheres as a universally applicable material for efficient and fast enrichment of peptides and proteins.