Glycosylation has significant effects on protein function and cell metastasis which are important in cancer progression. It is of great interest to identify site-specific glycosylation in search of potential cancer biomarkers. However, the abundance of glycopeptides is low compared to non-glycopeptides after trypsin digestion of serum samples, and the mass spectrometric signals of glycopeptides are often masked by coeluting non-glycopeptides due to low ionization efficiency. Selective enrichment of glycopeptides from complex serum samples is essential for mass spectrometry (MS)-based analysis. Herein, a strategy has been optimized using LCA enrichment to improve the identification of core-fucosylation (CF) sites in serum of pancreatic cancer patients. The optimized strategy was then applied to analyze CF glycopeptide sites in 13 sets of serum samples from pancreatic cancer, chronic pancreatitis, healthy controls and a standard reference. In total, 630 core-fucosylation sites were identified from 322 CF proteins in pancreatic cancer patient serum using an Orbitrap Elite mass spectrometer. Further data analysis revealed that 8 CF peptides exhibited a significant difference between pancreatic cancer and other controls, which may be potential diagnostic biomarkers for pancreatic cancer.