Objective: The objective of this study was to determine the novice trainee’s learning curve for oocytes retrieval procedure through assessment of oocytes retrieval efficiency, operative time and other operative characteristics. Methods: This retrospective cohort study included 200 consecutive patients undergoing transvaginal ultrasound guided oocytes retrieval procedure. Those patients underwent oocyte retrieval procedure by a single operator and one experienced supervisor. Their clinical data, including demographic data, ovarian stimulation cycle information, surgical procedure, and laboratory data were collected over 3 months. CUSUM analyses based on the operative time were performed to determine the learning curve. Results: The mean operative time was 10.10 min. Based on the CUSUM plot of operative time, the learning curve can be divided into three separated phases, phase 1 (case 1 to case 49) was learning phase, phase 2 (case 50 to case 130) was acquisition phase, and phase 3 (case 131 to case 200) was proficiency phase. The operative time was significantly shortened from phase 1 to phase 3 (phase 1, 13.37 ± 4.83min; phase 2, 10.21 ± 3.30 min; phase 3, 7.67 ± 3.24 min, P < 0.001). The oocyte retrieval efficiency was also notably improved from phase 1 to phase 3 (78.2% to 100% based on method 1 to determine oocyte retrieval efficiency, and 104.2% to 121.1% based on method 2 to determine oocyte retrieval efficiency). The retrieved oocytes number, the fertilization rate, clinical pregnancy rate among the three phases showed no significant differences. No patients had severe adverse events. As determined by multiple linear regression, learning phase is the only independent predictor of oocyte retrieval efficiency. Conclusion: Trainees practice transvaginal ultrasound guided oocytes retrieval are expected to achieve a stabilized procedure over consecutive training cases, with acquisition of the skills at 49 cases, and proficiency at 130 cases. Cumulative operative experience can improve the operative time and oocytes retrieval efficiency, but showed minimal influence on retrieved oocytes number and reproductive outcomes.