Gestational diabetes mellitus (GDM) is a frequent medical condition during pregnancy. Early diagnosis and treatment of GDM are crucial for both the mother and the baby. In the present study, we aimed to identify specific biomarkers to assist in the early detection of GDM and give some clues to the possible causes of GDM by comparing serum peptide profile differences between GDM patients and healthy controls. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used in combination with weak cation exchange magnetic bead (WCX-MB). Levels of four peptides (4418.9, 2219.7, 2211.5, and 1533.4 Da) were significantly different. Interestingly, three of them (4418.9, 2211.5, and 1533.4 Da) were identified when GDM patients with two degrees of glucose intolerance were compared. Additionally, peptides 2211.5 and 1533.4 Da showed a decreasing trend as glucose intolerance increased, while peptide 4418.9 Da exhibited the reverse tendency. In conclusion, our study provides novel insights into the altered serum peptide profile of GDM patients. The specific candidate biomarkers may contribute to the development of GDM.