Background: The effect of elevated levels of serum progesterone (P 4 ) and estradiol (E 2 ) on the day of human chorionic gonadotropin and their cut-off value on in vitro fertilization (IVF) outcomes is still not clear. Aims: The aim was to evaluate the association between serum P 4 , E 2 and progesterone/estradiol ratio (P 4 /E 2 ) on pregnancy outcome in IVF/intracytoplasmic sperm injection (ICSI) cycles with long agonist protocol. Setting and Design: Retrospective, single center, cohort study. Materials and Methods: A review of complete data of 544 women undergoing fresh IVF/ICSI cycles (539 cycles) with long agonist protocol from January 2012 to February 2014 was done. Data were stratified into Three groups according to the number of oocytes retrieved: low (≤4 oocytes obtained), intermediate (5-19 oocytes obtained), and high ovarian response (≥20 oocytes obtained). Statistical Analysis: Fishers exact test/Chi-square was carried for comparing categorical data. Receiver operating characteristics analysis was performed to determine the cut-off value for P 4 and P 4 /E 2 detrimental for pregnancy. Results: A negative association was observed between pregnancy rate (PR) and serum P 4 and P 4 /E 2 levels with no effect on fertilization and cleavage rate. The overall cut-off value of serum P 4 and P 4 /E 2 ratio detrimental for pregnancy was found to be 1.075 and ≥0.35, respectively. Different P 4 threshold according to the ovarian responders were calculated, 1.075 for intermediate and 1.275 for high responders. Serum E 2 levels were not found to be significantly associated with PR. Conclusion: Serum P 4 levels and P 4 /E 2 ratio are a significant predictor for pregnancy outcome without affecting cleavage and fertilization rate while serum estradiol levels do not seem to affect PR.