Objective. The purpose of this study was to screen serum proteins for biomarkers of gestational diabetes mellitus (GDM) and to investigate its pathogenesis by analyzing the differences in serum proteomics between pregnant women with GDM and healthy pregnant women. Methods. Patients who were admitted to the First Affiliated Hospital of Fujian Medical University from June 2019 to January 2020 were included. According to the medical history and the results of the 75 g oral glucose tolerance test (OGTT), they were divided into the normal pregnant women group and GDM pregnant women group. The serum of two groups of patients was collected. High performance liquid chromatography-mass spectrometry was used to identify differentially expressed serum proteins between pregnant women with GDM and healthy pregnant women, and bioinformatics analysis was then performed on the identified proteins. Results. A total of 1152 quantifiable proteins were detected; among them, 15 were upregulated in serum of GDM pregnant women, while 26 were downregulated. The subsequent parallel reaction monitoring (PRM) assay validated the expression levels of 12 out of 41 differentially expressed proteins. Moreover, bioinformatics analysis revealed that the differentially expressed proteins are involved in multiple biological processes and signaling pathways related to the lipid metabolism, glycan degradation, immune response, and platelet aggregation. Conclusions. This study identified 41 serum proteins with differential expression between pregnant women with GDM and healthy pregnant women, providing new candidate molecules for elucidating GDM pathogenesis and screening therapeutic targets.