Background/Objectives: Maternal amino acid intake and its biological value may influence glucose regulation and insulin sensitivity, impacting the risk of developing gestational diabetes mellitus (GDM). This study aimed to evaluate the association between amino acid intake from maternal diet before and during pregnancy and the risk of GDM. Methods: This study is part of the ongoing BORN2020 epidemiological Greek cohort. A validated semi-quantitative Food Frequency Questionnaire (FFQ) was used. Amino acid intakes were quantified from the FFQ responses. A multinomial logistic regression model, with adjustments made for maternal characteristics, lifestyle habits, and pregnancy-specific factors, was used. Results: A total of 797 pregnant women were recruited, of which 14.7% developed GDM. Higher cysteine intake during pregnancy was associated with an increase in GDM risk (adjusted odds ratio [aOR]: 5.75; 95% confidence interval [CI]: 1.42–23.46), corresponding to a 476% increase in risk. Additionally, higher intakes of aspartic acid (aOR: 1.32; 95% CI: 1.05–1.66), isoleucine (aOR: 1.48; 95% CI: 1.03–2.14), phenylalanine (aOR: 1.6; 95% CI: 1.04–2.45), and threonine (aOR: 1.56; 95% CI: 1.0–2.43) during pregnancy were also associated with increased GDM risk. Furthermore, total essential amino acid (EAA) (aOR: 1.04; 95% CI: 1.0–1.09) and non-essential amino acid (NEAA) (aOR: 1.05; 95% CI: 1.0–1.1) intakes during pregnancy were also linked to an increased risk of GDM. A secondary dose–response analysis affected by timing of assessment revealed that higher intake levels of specific amino acids showed a more pronounced risk. Conclusions: Optimizing the balance of certain amino acids during pregnancy may guide personalized nutritional interventions to mitigate GDM risk.