It is becoming a public health issue to predict which expectant women will develop gestational diabetes mellitus (GDM). The goal of this case control research is to investigate the role of maternal oxidative stress levels in the first, second, and third trimesters, as well as other factors, in the development of gestational diabetes mellitus (GDM). Methods Between October and December 2021, 142 women participated in this research. The 101 GDM patients were split into three groups based on their gestation (T1, T2, and T3), and 41 healthy pregnant women were chosen as the comparison group. TAS and TOS levels of oxidative stress and XO were calculated using a Spectrophotometer for colorimetric techniques; fasting and random sugar levels, as well as HbA1c, were evaluated. Results Afamin levels were significantly greater in women with GDM in all trimester (T1=51.63 ± 3.86, T2=57.39 ± 3.17, T3=64.22 ± 3.67 ng/mL) in comparison with control (45.93 ± 2.26) with statistical significance (P ≤ 0.01). Afamin levels were higher significantly in T3>T2>T1.The levels of oxidative stress were higher significantly in women with GDM in all trimester (T1=73.79 ± 18.43, T2=81.28 ± 18.06, T3=96.70 ± 20.69) in comparison with control (37.28 ± 18.95) with statistical significance (P = 0.000). Oxidative stress levels were higher significantly in T3>T2>T1. The measured TOS and XO levels were  higher in all GDM groups compared with control (p=0.000) and high significant between patient groups (T3>T2>T1), while high significant of TAS in GDM than control but (T3