Gestational diabetes mellitus (GDM) is a form of hyperglycemia that arises during pregnancy and is associated with increased maternal and fetal risks. The current diagnosis relies on an oral glucose tolerance test performed between 24 and 28 weeks of gestation, limiting the window for early intervention. This study aimed to analyze serum proteins as potential biomarkers for early diagnosis and monitoring of GDM in Mexican women. A cohort of 200 pregnant women was enrolled during their first trimester, with serum samples collected in each trimester. A GDM diagnosis was confirmed in the second trimester according to the international guidelines. After applying inclusion and exclusion criteria, 19 women with GDM and 15 women without GDM were included in the final analysis. Nine candidate proteins (ADIPOQ, PAPP-A, PAPP-A2, IGFBP-5, AFM, SHBG, FABP4, RBP4, and RETN) were measured in the first-trimester serum using a targeted antibody array. Multivariate and linear regression analyses identified SHBG, IGFBP-5, and FABP4 as promising biomarkers for early GDM detection. Quantification across all trimesters was performed using ELISA kits. The receiver operating characteristic (ROC) curve analysis showed that FABP4 exhibited the highest sensitivity and specificity for discriminating GDM cases during the first trimester. However, its diagnostic performance decreased as pregnancy progressed, limiting its utility for monitoring disease progression. These findings support the potential of FABP4 as a promising candidate for early GDM diagnosis in Mexican women.