Background: Hormone Replacement Therapy-Frozen Embryo Transfer (HRT-FET) cycles are widely used in assisted reproductive technology, yet the optimal midluteal estradiol (E2) level on embryo transfer day remains unclear. Aim: To evaluate whether serum E2 levels on the day of embryo transfer influence clinical outcomes in HRT-FET cycles. Settings and Design: This prospective observational study was conducted from July to December 2024 at a tertiary reproductive center in India. Materials and Methods: Fifty infertile women were allocated to either Day 3 (n=14) & Day 5 (n=36) embryo transfers. After downregulation, endometrial preparation was performed with oral estradiol valerate, followed by daily intramuscular progesterone. Participants were stratified into low (≤187 pg/mL) or high (>187 pg/mL) E2 groups on embryo transfer day. Clinical pregnancy rate (CPR) and implantation rate (IR) were measured. Statistical Analysis Used: Data analysis was conducted using SPSS version 29. Groups were compared using independent t-tests, Mann–Whitney U, and chi-square tests. Binary logistic regression assessed associations between E2 levels and other parameters. A p-value <0.05 was considered significant. Results: In Day 5 transfers, the high E2 group had higher CPR (46.6% vs. 28.57%) and IR (28% vs. 15%), though differences were not statistically significant. Serum E2 was negatively correlated with BMI (p=0.044) and positively correlated with endometrial thickness (p=0.012). E2 showed a significant association with CPR (p=0.039). Conclusions: This study emphasizes that the higher midluteal estradiol levels on embryo transfer day may improve pregnancy outcomes in HRT-FET cycles, especially in Day 5 transfers.