Aim The aim of this study was to assess the role of total serum estradiol on the day of injection HCG, estradiol per mature follicle, and estradiol per oocytes retrieved (OR) on clinical pregnancy rate (CPR) and oocyte/embryo quality in assisted reproduction. Materials and Methods A retrospective review of 342 in vitro fertilization cycles with normal ovarian reserve in women who underwent long GnRH agonist protocol was included. The outcomes assessed are number of OR, number of mature oocytes (MO), number of oocytes fertilized (FO), fertilization rate, number of embryos cleaved (EC), cleavage rate (CR), number of Grade I embryos (E), number of cryopreserved embryos (CPE), and CPR. The Estradiol/follicle ratio (E2/fol) was defined as estradiol level per mature follicle [14 mm in diameter. Estradiol/ oocyte (E2/O) ratio was defined as estradiol level per OR. These two ratios were categorized by the 25th percentile into four groups. Results A positive correlation was seen between E2/fol and OR (r = .334, p value = .0001), MO (r = .335, p value = .0001), FO (r = .222, p value = .002), and CPE (r = .289, p value = .0001). Increased CPR was seen in Group C (E2/ fol = 200-299.99) compared to Group A, B, and D (p value = .033). With E2/O ratio, negative correlation was seen between E2/O and OR (r = -.281, p value = .002), MO (r = -.296, p value = .008), FO (r = -.220, p value = .003), EC (r = -.211, p value = .004), Grade 1 embryo (r = -.216, p value = .001), and CPE (r = -.206, p value = .005). No difference in FR, CR, or CPR was seen. No difference was seen in CPR with total serum estradiol. Conclusions In conclusion, serum estradiol is an important determinant of IVF success. While total serum estradiol does not exert any positive or negative influence on IVF outcome, estradiol per mature follicle and retrieved oocytes do have an impact. Pregnancy rate is better when E2/fol is between 200 and 299.99 pg/ml. Also, increasing serum E2/fol positively correlates with better oocytes and embryo quality. In contrast, E2/O negatively correlates with oocytes and embryo quality parameters.