Serum peptide profiling is a promising approach for classification of cancer versus noncancer samples. In this study, we aimed to search for discriminating peptide patterns in serum samples between lung cancer patients and healthy controls. The magnetic beads-based weak cation-exchange chromatography followed by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used in this study to identify patients with lung cancer. In total, serum samples from 64 lung cancer patients (32 for training set and 32 for testing set), 64 healthy controls (32 for training set and 32 for testing set), and 10 COPD patients (for disease control) were analyzed in this study. The mass spectra data analyzed with ClinProTools software was used to distinguish between cancer patients and healthy individuals based on three different algorithm models (GA, SNN, and QC). In the training set, patients with lung cancer could be identified with the mean sensitivity of 98.9% and specificity of100%. Similar results could be obtained from testing set, showing 87% sensitivity and 84.8% specificity. Screening for serum peptide patterns using MALDI-TOF MS showed high sensitivity and specificity in identifying patients with lung cancer.