Objective: This study aimed to find the association between alanine transaminase-toaspartate aminotransferase ratio (ALT/AST) and the incidence of gestational diabetes mellitus (GDM). Methods: A total of 1128 pregnant women were included in this prospective, double-center, observational cohort study. ALT, AST and total bilirubin (TBil) were tested during 6-12 weeks of gestation and 75-g oral glucose tolerance test (OGTT) was conducted during 24-28 weeks of gestation to screen GDM. The association between ALT/AST and glucose concentration during OGTT was analyzed by linear regression model. The OR with 95% CI for incidence of GDM associated with ALT/AST was estimated by binary logistic regression. The discriminatory values of ALT/AST and triglyceride/high-density lipoprotein cholesterol (TG/HDL-C) for GDM were calculated by the area under the receiver operating characteristic curve (ROC-AUC). Results: The incidence of GDM was 22.07% (249/1128). ALT/AST was higher in GDM group than in NGT group (0.92 [0.75, 1.18] vs 0.80[0.65, 1.02], P <0.001). ALT/AST had positive correlations with fasting blood glucose, 1-hour and 2-hour blood glucose concentration during OGTT (0.089 [95% CI: 0.034, 0.163], 0.176 [95% CI: 0.052, 0.104], and 0.115 [95% CI: 0.199, 0.609], respectively). The OR of ALT/AST for incidence of GDM was 1.603 (95% CI:1.097, 2.344). The ROC-AUC of ALT/AST and TG/HDL-C reached 0.615 (95% CI: 0.575, 0.655) and 0.619 (95% CI: 0.580, 0.659), respectively. Conclusion: ALT/AST in early pregnancy was an independent risk factor of GDM. The predictive ability of ALT/AST was similar to TG/HDL-C for GDM.