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Peptidomics research is of great significance for discovering potential biomarkers and monitoring human diseases. As a kind of common clinical biofluid, saliva known for its noninvasive collection and easy accessibility has been widely used in peptidomics research. In this article, we combined immobilized metal ions affinity chromotography (IMAC) with mesoporous material and proposed the copper ion doped magnetic mesoporous silica material (denoted as Fe3O4@mSiO2-Cu2+) which had a large surface area of 221 m2 g−1 and pore volume of 0.20 cm3 g−1. By immobilizing copper ions onto the mesopore walls, the standard peptide Angiotensin II could be identified in an extremely low concentration of 0.1 fmol μl−1 and in a mass ratio of 1:500 (Angiotensin II:BSA, m/m), which indicated significant sensitivity and a great size-exclusive ability. In addition, the introduction of polydopamine (PDA) made Fe3O4@mSiO2-Cu2+ more hydrophilic and biocompatible which could improve the profiling of endogenous peptides in bio-sample. Finally, 131 endogenous peptides were identified in human saliva after enrichment with Fe3O4@mSiO2-Cu2+. Therefore, Fe3O4@mSiO2-Cu2+ nanoparticles provided a promising candidate protocol for biomarker discovery.