Objective To determine the median effective dose (ED50) and 95% effective dose (ED95) of alfentanil combined with ciprofol in painless oocyte retrieval. Methods Patients, aged 21‒40 years, with BMI 18.5‒28.3 kg/m2 and American Society of Anesthesiologists (ASA) grade Ⅰ or Ⅱ, who underwent painless oocyte retrieval were enrolled. The initial dose of alfentanil was 4 μg/kg, ciprofol was 0.4 mg/kg, and the dose of alfentanil for the next patient was determined using the modified sequential method. The incidence of adverse reactions (hypotension, hypoxemia, bradycardia, dizziness, nausea and vomiting) was recorded. Probit regression analysis was used to calculate the ED50 and ED95 of alfentanil, and the corresponding 95% confidence interval (CI). Results The ED50 and ED95 of alfentanil combined with ciprofol in painless oocyte retrieval was 3.8 (3.2, 4.2) μg/kg and 4.6 (4.2, 9.7) μg/kg, respectively. There were four cases of hypotension, three cases of hypoxemia, and one case of dizziness, without nausea and vomiting, and bradycardia. Conclusion The ED50 and ED95 of alfentanil combined with ciprofol (0.4 mg/kg) in painless oocyte retrieval are 3.8 μg/kg and 4.6 μg/kg.