Objective: To explore the effect of different fertilization time after human chorionic gonadotropin (HCG) injection on the outcome of fertilization in vitro fertilization-embryo transfer (IVF-ET). Methods: One thousand one hundred and forty IVF-ET cycles from January 2016 to August 2018 were analyzed retrospectively. According to the different fertilization time after injection of HCG divided into four groups: Group A (38.0 h～39.0 h), Group B (39.1 h～40.0 h), Group C (40.1 h～41.0 h), and Group D (41.1 h～42.0 h). The normal fertilization rate, the normal cleavage rate, the embryo utilization rate, the high-quality embryo rate, the clinical pregnancy rate, the implantation rate, and the spontaneous abortion rate were analyzed among the groups. Then we investigated the effect of different promotion methods on the outcome of fertilization during the optimal fertilization time. Results: There was no significant difference in 2PN cleavage rate, available embryo rate, clinical pregnancy rate, implantation rate and abortion rate among the four groups (P>0.05). The high-quality embryo rate in Group D (44.6％) was the highest, and was significantly different among the four groups (P<0.05). The normal fertilization rate in Group D (71.6％) was the highest, and was significantly different among the four groups (P<0.05). The normal fertilization rate (78.1％) of antagonist group was significantly higher than other groups (P<0.05). Conclusion: The different fertilization time after HCG injection have effects on high-quality embryo rate and normal fertilization rate of patients in IVF-ET. The appropriate fertilization time of patients in IVF-ET was 41 h～42 h after HCG injection in our reproductive center, improved the clinical pregnancy rate and reduced the early abortion rate. The GnRH-ant protocol is superior to other protocol in IVF-ET.