Polycystic ovary syndrome (PCOS) is a prevalent endocrine disorder characterised by insulin resistance, oxidative stress, and dyslipidaemia, contributing to long-term metabolic and cardiovascular complications. Despite available therapies, effective and sustainable alternatives remain limited, highlighting the need for novel adjunct interventions. This study investigated the effects of biotin supplementation on glucose metabolism, antioxidant status, and lipid profile in letrozole-induced PCOS female Wistar rats. A controlled experimental design was adopted, with 30 rats randomly assigned to five groups (n = 6): control, letrozole-induced PCOS, letrozole + metformin, letrozole + low-dose biotin (100 mg/kg), and letrozole + high-dose biotin (150 mg/kg). Treatments were administered orally for 15–21 days. Biochemical parameters were analysed using standard laboratory methods, and data were expressed as mean ± SD. One-way ANOVA was used for statistical analysis, with p < 0.05 considered significant. Letrozole induction significantly increased blood glucose (95.91 ± 2.35 mg/dl) and reduced antioxidant enzyme activity compared with the control (p < 0.05). High-dose biotin significantly reduced glucose levels (85.55 ± 2.46 mg/dl) and improved catalase, SOD, GSH, and GST activities compared with the PCOS group (p < 0.05), though metformin achieved superior normalisation (75.01 ± 2.38 mg/dl). Biotin supplementation also improved lipid abnormalities, with high-dose biotin reducing LDL (27.89 ± 1.00 mg/dl) and increasing HDL (155.59 ± 1.74 mg/dl) relative to letrozole (p < 0.05). In conclusion, biotin supplementation, particularly at high doses, partially ameliorated hyperglycaemia, oxidative stress, and dyslipidaemia in PCOS-induced rats. Biotin may serve as a supportive therapeutic agent in PCOS management. Further studies are recommended to determine optimal dosing, underlying mechanisms, and long-term clinical relevance.