Background: The purpose of the study was to determine cut-off values for ovarian volume (OV) and follicle number per ovary (FNPO) in Indian women with polycystic ovary syndrome (PCOS). Methods: Eighty six PCOS women (Rotterdam criteria) and forty five matched ovulatory and normo-androgenic women were recruited. A detailed 2D and 3D trans-vaginal scan was carried out in early follicular phase (D2-D5) in all patients. Ovarian volume, follicle number per ovary, stromal volume, vascularization index (VI), vascularization flow index (VFI) and flow index (FI) were measured in PCOS and controls. Mann-Whitney test and logistic analysis using PROC LOGISTIC function of SAS ® (Version 9.3) were used to calculate the best cut-offs for the diagnosis of PCOS. Results: Mean ovarian volume was 13.7±5.89 and 5.06±2.44 (p<0.0001), FNPO was 19.18±6.89 and 7.13±3.51 (p<0.0001) in PCOS and controls, respectively. The cut-offs for the diagnosis of PCOS were 2D OV=6.15 cm³, 2D FNPO=12. By 3D scan, OV=7 cm³, FNPO=10, stromal volume=6 cm³, VI=4.546, VFI=2.925 and FI=19.266. Youden’s Index (To select optimal predicted probability cut-off) was the highest for 2D FNPO (0.88786). 2D FNPO showed the highest specificity and sensitivity (AUC), 0.95238 and 0.93548, for the diagnostic accuracy of PCOS. Conclusion: 2D and 3D trans-vaginal scans are equally accurate for assessment of ovarian morphology. FNPO has better diagnostic accuracy for PCOS compared to ovarian volume. Cut-off for FNPO and OV in Indian PCOS women is 12 and 6.15 cm³ by 2D, 10 and 7 cm³ by 3D trans-vaginal scan.