With the advent of post-genomic era, it is an important task to study the expression, modification and interaction of the total proteins in cells, tissue and organs for proteomics in biological organism. The protein phosphorylation is one of the most popular mechanisms in signal transduction and enzyme modulation. It is estimated that about 2% of the total genes encoded 500 kinases and 100 phosphatases in human genome. As the key point of expression modulation in prokaryotic and eukaryotic cells, the protein phosphorylation and dephosphorylation may help to reveal the status of the life system at the molecular level. The phosphorylation in proteome remains challenging for functional genomics. In the present review, some new advances such as identification and characterization of protein phosphorylation in proteome are summarized.