Background: Polycystic ovarian syndrome (PCOS) is a prevalent endocrine disorder characterized by hormonal imbalance and metabolic dysfunction, often diagnosed through clinical, biochemical, and radiological markers. Transvaginal ultrasound follicle count and serum anti-Müllerian hormone (AMH) levels have been increasingly used as key diagnostic tools for defining polycystic ovaries. However, establishing threshold values for these parameters to differentiate PCOS from other ovarian conditions remains challenging. Methodology: The study design was case control study took place at Hayatabad Medical complex Peshawar. A total of 150 women diagnosed with PCOS were compared with 100 controls. Follicle count was determined through transvaginal ultrasound, while serum AMH levels were measured using enzyme-linked immunosorbent assay (ELISA). Statistical analyses, including correlation and regression, were performed to explore associations between variables, and receiver operating characteristic (ROC) curves were used to evaluate diagnostic performance. Results: The PCOS group exhibited significantly higher follicle counts (mean: 25.4 ± 7.2) and AMH levels ‘(6.2 ± 1.7 ng/mL) compared to controls (12.6 ± 3.4 follicles; AMH: 2.1 ± 0.8)’. A moderate positive correlation (correlation coefficient = 0.65, p-value < 0.001) was identified between follicle count and AMH levels in the PCOS group. The combination of a follicle count ≥20 and AMH levels ≥4.5 ng/mL showed excellent diagnostic performance, with an ‘area under the curve (AUC)’ of 0.88, a sensitivity of 90%, and a ‘specificity of 80’%. Conclusion: Both follicle count and AMH levels are reliable markers for diagnosing PCOS. The combination of these two parameters offers greater diagnostic precision than either alone, supporting the need for standardized thresholds in clinical practice.