Protein phosphorylation is one of the most important posttranslational modifications in mammalian cells. It regulates numerous biological processes, including cell proliferation, differentiation, metabolism, communication, and signal transduction. Global analysis of protein phosphorylation is very significant for exploring these critical processes. In this study, Fe3+immobilized magnetic nanoparticles (Fe3+-IMAN) with an average diameter of 15 nm were synthesized and applied to enrich phosphopeptides. Compared with commercial microscale IMAC beads, Fe3+-IMAN has a larger surface area and better dispersibility in buffer solutions which improved the specific interaction with phosphopeptides. Using tryptic digests of the phosphoprotein α-casein as a model sample, the number and signal-to-noise ratios of the phosphopeptides identified by matrixassisted laser desorption/ionization time-of-flight mass spectrometry (MALDITOFMS) following Fe3+-IMAN enrichment greatly increased relative to results obtained with direct MALDITOFMS analysis. The lowest detectable concentration is 5 x10 -11 M for 100 μL of pure standard phosphopeptide (FLTEpYVATR) following Fe3+-IMAN enrichment. We presented a phosphopeptide enrichment scheme using simple Fe3+-IMAN and also a combined approach of strong cation exchange chromatography and Fe3+-IMAN for phosphoproteome analysis of the plasma membrane of mouse liver. In total, 217 unique phosphorylation sites corresponding to 158 phosphoproteins were identified by nano-LC-MS/MS. This efficient approach will be very useful in large-scale phosphoproteome analysis.