Gestational diabetes mellitus (GDM) is an abnormal state of glucose metabolism that first appears during pregnancy and poses a serious threat to maternal and infant health. In recent years, with the change of lifestyle and the update of diagnostic criteria, the incidence of GDM has increased year by year. Blood samples from pregnant women diagnosed with gestational diabetes were collected, concentrations of IGFBP5 protein molecules were measured by enzyme-linked immunosorbent assay (ELISA), and thyroid stimulating hormone (TSH) levels were measured by radioimmunoassay. Relevant clinical data were collected to exclude other variables that might affect the results. Correlation analysis showed that IGFBP5 protein concentration was negatively correlated with blood glucose level, but positively correlated with TSH level. Multiple regression analysis further revealed that IGFBP5 protein molecules and TSH levels are important factors affecting the development of gestational diabetes mellitus. This study confirms the diagnostic potential of the IGFBP5 protein molecule in gestational diabetes and reveals the complex effects of its interaction with thyroid stimulating hormone on glucose metabolism. The decrease of IGFBP5 protein concentration and thyroid stimulating hormone level may be the markers of the development of GDM. Therefore, the detection of IGFBP5 protein molecules and thyroid stimulating hormone levels is expected to provide new ideas and methods for the early diagnosis and treatment of GDM.