The goal was to analyze serums of GDM patients and healthy pregnant women using HPLC-MS and preliminarily screen differential metabolites by metabolomics. Sixty pregnant women who underwent elective cesarean section at term in Dongguan Dalang Hospital from January 2023 to April 2023 were selected and divided into the GDM group and healthy pregnancy group. Pre-pregnancy and pregnancy examination information, such as age, BMI, OGTT results, triglyceride, total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, and other clinical data were col-lected for statistical analysis. Non-targeted metabolomics of serum from 30 GDM patients and 30 healthy pregnant women were studied by HPLC-MS, and different ions were searched. The structures of differential metabolites were identified by HMDB database. The metabolic pathways of differential metabolites were analyzed by KEGG database. The OGTT result, pCO2, pO2, HCO3, BE, Apgar score, and bilirubin levels in the GDM group were higher than those in the healthy pregnancy group (p < 0.05). However, there were no significant differences in age, triglyceride, total cholesterol, newborn birth weight, newborn birth blood glucose, and blood gas pH between the two groups (all p > 0.05). Using p < 0.05 as the screening standard, 55 differential metabolites were identified in serum, mainly including fatty acyl, carboxylic acids and their derivatives, steroids and their derivatives, ketoacids and their derivatives, and pyrimidine nucleosides, etc., all of which were up-regulated or down-regulated to varying degrees. The 55 metabolites were mainly involved in the metabolism of pyrimidine, pyruvate, alanine, aspartic acid, glutamic acid, and arachidonic acid, glycolysis, and biosynthesis of unsaturated fatty acids. The discovery of these metabolites provides a theoretical basis for an indepth understanding of GDM pathogenesis. Non-targeted metabonomics analysis of blood metabonomics research technology has shown great potential value in the early diagnosis of obstetric diseases and the study of disease mechanisms.