Core fucosylation, a special type of -linked glycosylation, is important in tumor proliferation, invasion, metastatic potential, and therapy resistance. However, the core-fucosylated glycoproteome has not been extensively profiled due to the low abundance and poor ionization efficiency of glycosylated peptides. Here, a "one-step" strategy has been described for protein core-fucosylation characterization in biological samples. Core-fucosylated peptides can be selectively labeled with a glycosylated probe, which is linked with a temperature-sensitive poly(-isopropylacrylamide) (PNIPAM) polymer, by mutant endoglycosidase (EndoF3-D165A). The labeled probe can be further removed by wild-type endoglycosidase (EndoF3) in a traceless manner for mass spectrometry (MS) analysis. The feasibility and effectiveness of the "one-step" strategy are evaluated in bovine serum albumin (BSA) spiked with standard core-fucosylated peptides, H1299, and Jurkat cell lines. The "one-step" strategy is then employed to characterize core-fucosylated sites in human lung adenocarcinoma, resulting in the identification of 2494 core-fucosylated sites distributed on 1176 glycoproteins. Further data analysis reveals that 196 core-fucosylated sites are significantly upregulated in tumors, which may serve as potential drug development targets or diagnostic biomarkers. Together, this "one-step" strategy has great potential for use in global and in-depth analysis of the core-fucosylated glycoproteome to promote its mechanism research.