Abstract: Protein phosphorylation is one of the most common post-translational modifications(PTMs)in various organisms, which plays critical roles in the regulation of intracellular biological processes such as cell proliferation, signal transduction, metabolismics and tumorigenesis. The understanding of phosphoproteomics is playing a great role in biomarker discovery, auxiliary treatment, as well as biological process exploration. However, the identification and characterization of phosphoproteins remain challenging due to their low abundance and the signal suppression of nonphosphorylated peptides. Thus, extraction of phosphorylated polypeptides from complex mixtures is a critical step in the success of phosphoproteomics experiments. Some strategies have been explored to promote enrichment specificity, detection sensitivity and enrichment capacity, such as design of new nanomaterials or combination of a variety of analytical methods. This review places special emphasis on the recent four-year advances for enrichment strategies that have been used for phosphoproteomics and summarize the application of phosphoproteomics in disease research.