Protein glycosylation, mainly divided into N-glycosylation and O-glycosylation, is the most prevalent and complex protein posttranslational modification. Mapping of glycosylation sites where glycans attached to proteins is the prerequisite to un-cover the biological functions that glycans played in living cells. However, profiling of multiple glycosylations from a given biological sample is still a challenging issue in glycoscience. Here, a strategy integrates one-step enzymatic labeling and multi-steps release was developed to enable simultaneous profiling of N-glycosylation and truncated O-glycosylation by mass spec-trometry (MS) technology. A probe functionalized with temperature-sensitive polymer and a cleavable linker can be tagged to N-glyans and O-glycans by two substrate-specific sialyltransferases in a one-step manner. Without the need of additional biorthogonal reaction to introduce enrichment tag or resin, the labelled peptides can be directly precipitated out of the solu-tion in water bath to achieve enrichment (0 °C). Then, the captured glycpetides were released by endoglycosidase and UV356 irradiation sequentially and analyzed by MS. After searching database using different variable modifications, glyco-proteomes including N-glycosylation, core fucosylation and truncated mucin-type O-glycosylation can be profiled successfully.