BACKGROUND: The effect of elevated levels of serum progesterone (P4) and estradiol (E2) 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 P4, E2 and progesterone/estradiol ratio (P4/E2) 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 P4 and P4/E2 detrimental for pregnancy. RESULTS: A negative association was observed between pregnancy rate (PR) and serum P4 and P4/E2 levels with no effect on fertilization and cleavage rate. The overall cut-off value of serum P4 and P4/E2 ratio detrimental for pregnancy was found to be 1.075 and ≥0.35, respectively. Different P4 threshold according to the ovarian responders were calculated, 1.075 for intermediate and 1.275 for high responders. Serum E2 levels were not found to be significantly associated with PR. CONCLUSION: Serum P4 levels and P4/E2 ratio are a significant predictor for pregnancy outcome without affecting cleavage and fertilization rate while serum estradiol levels do not seem to affect PR.