Protein N-phosphorylation enrichment is faced with great difficulties due to the intrinsic instability of the N-P bond, which has seriously hindered its biological function unraveling. Current methods for N-phosphopeptides enrichment are challenging to fulfil high selectivity for all types of N-phosphorylation with minimum contamination. To address this, we reported the first affinity peptide functionalized magnetic beads for N-phosphopeptides enrichment under neutral aqueous solution. The affinity peptide, capable of binding the N-PO3 group specifically, was identified using phage display technology based on a delicate target. We demonstrated robust enrichment capacity of the peptide-modified magnetic beads across diverse biological samples, from prokaryotes to eukaryotes, as well as various organelles and tissues. Accordingly, 262 and 2307 N-phosphorylation sites were identified from E.coli and HeLa cells, which demonstrated the enhanced enrichment capability and greatly expanded the scale of N-phosphoproteome databases. Furthermore, we found that N-phosphorylation was highly enriched in the nucleus and played a vital role in regulating processes like DNA repair. Given the high efficiency and quantitative reproducibility of the method, it was found that N-phosphorylation level for kinases and proteins involved in regulation of metabolism and microtubules varied during the progression of Alzheimer's disease. Our work expands the tools library for N-phosphorylated proteome, and provides critical insights into the roles of N-phosphorylated proteins in physiology and pathology processes.