In addition to O-phosphorylation, protein N-phosphorylation was proven to play important roles in multiple biological processes. Although affinity methods were developed for the enrichment of N-phosphorylation peptides in cells, it was still difficult to enrich N-phosphorylation peptides in tissue due to its complexity. In this study, we established a workflow for the identification of N-phosphorylation peptides in mouse brain tissue by direct enrichment in high concentration urea. In total, 989 N-phosphorylation sites were obtained using 0.5 M urea as enrichment buffer. Among all identified N-phosphorylation sites, the localization probability over 0.75 was as high as 80%, suggesting the reliability of the method. Furthermore, the sequence motif analysis and gene ontology analysis results showed a good match to previous studies. The method was successfully used for N-phosphorylation analysis of mouse hippocampus from Alzheimer's disease model, and 533 N-phosphorylation sites were identified in 5 × FAD mouse hippocampus tissue. Biological process analysis results showed that "brain development", "cellular response to reactive oxygen species", "microtubule cytoskeleton organization", and "peptidyl-serine phosphorylation" were especially enriched in 5 × FAD mouse. It is suggested that N-phosphorylation may be related to Alzheimer's disease in these aspects.