Lung cancer (LC) is one of the leading causes of cancer deaths worldwide and approximately 3 million individuals die from this disease each year. Therefore, detecting LC at its early stages is very important to achieve decreased LC mortality. While gene expression profiling has been successfully used to classify various tumors and assess tumor stages, the gene-based prediction for LC is not yet entirely dependable. In serum, glycosylation is one of the most common post-translational modifications, and glycoproteins play a core role in a diversity of biological processes and pathogenesis. The development of an analytical approach for the study of variations of human serum glycoproteins has been limited by the structural heterogeneity of the post-translational modifications and the complexity of glycomics. Thus, in this report we present a strategy of using Concavanalin A (Con A) as an affinity agent combined with twodimensional electrophoresis (2-DE) and nanoLC ESI-MS/MS to enrich and characterize the N-linked glycoproteins that are the most common glycosylation motifs in serum. By comparison of serum samples between LC patients and the healthy, we found that 8 glycoproteins were significantly up regulated in LC serum and 3 glycoproteins were down regulated. The useful information related to LC was discovered while we investigated changes of glycosylation in LC serum. The results suggested that the combination of proteomic techniques could be used for mining protein biomarkers for LC.