We developed a tumor extracellular O-GlcNAcylated proteome identification system, which involving removement of tumor cells by centrifugation, collection of extracellular O-GlcNAcylated proteome by immunoprecipitation using anti-O-GlcNAc antibodies and measurement of data by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The 89 proteins were identified to be overlapped with 3 distinct LUAD tissues and they were potential O-GlcNAcylated proteins.