Small Ubiquitin-like Modifier(SUMO) plays important roles in regulating many physiological activities of cells. Accurate identification and quantification of the SUMO substrates and modification sites at proteome scale is helpful for studying the molecular mechanisms of SUMO modification and its' influence on the functions of protein substrates. However, there is no method for the selective enrichment and efficient and accurate identification of wild-type SUMO modified peptides in various kinds of protein samples. In this work, a method for enrichment of endogenous SUMOlyated peptides based on anion chromatography and antibody was developed. Standard SUMO modified peptides library was synthesized and high efficient and accurate algorithms for the identification of SUMO modified peptides was developed by investigating their fragmentation patterns in mass spectrometer. The developed method was applied to the proteome-scale analysis of SUMO modified proteins and modification sites to the analysis of SUMO substrates and their modification sites in HeLa cells, which provides important technical support for disclosing the regulation networks of SUMO modification and studying the functions of SUMO modification in biological processes.