Analysis of protein phosphorylation remains a significant challenge due to the low abundance of phosphoproteins and the low stoichiometry of phosphorylation, which requires effective enrichment of phosphoproteins. Here we have developed superparamagnetic nanoparticles (NPs) whose surface is functionalized by multivalent ligand molecules that specifically bind to the phosphate groups on any phosphoproteins. These NPs enrich phosphoproteins from complex cell and tissue lysates with high specificity as confirmed by SDS-PAGE analysis with a phosphoprotein-specific stain and mass spectrometry analysis of the enriched phosphoproteins. This method enables universal and effective capture, enrichment, and detection of intact phosphoproteins toward a comprehensive analysis of the phosphoproteome.