Background: Anti-Müllerian hormone (AMH) has been recommended as the primary surveillance modality for evaluation of ovarian insufficiency in females. Patients with Ewing sarcoma receive VDC-IE chemotherapy which is estimated high risk of gonadotoxic injury. Previous studies have shown that only a small part of female patients with Ewing sarcoma show signs of ovarian recovery during long-term follow-up, similar to those treated with SCT or abdominal radiotherapy. Time to start preservation of fertility should be defined in this group of patients. Methods: In this observational study, we prospectively investigated AMH as a measure of ovarian function in female adolescents (less than 18 years old) with Ewing sarcoma who received initial chemotherapy in our hospital. Patients previously treated with systemic treatment were excluded. Serum levels of AMH were measured at the laboratory of Peking University People’s Hospital. Blood samples were collected at the first day of the initial two cycles of VDC-IE treatment, before local therapy including surgical resection and radial radiation, and at the end of chemotherapy. All statistical analyses were performed using SPSS 19.0 version. Results: A total of 53 female adolescents with Ewing sarcoma who received initial chemotherapy in our hospital were analyzed from February 1, 2021 to December 31, 2023, including 35 patients aging from 6 to 12 (group 1), 18 patients aging from 12 to 18 (group 2). 255 blood samples from unique time points were measured. The baseline mean AMH were 4.16 (90% CI, 1.88 – 9.42) ng/mL in all patients, 3.58 (90% CI, 1.40 – 6.91) ng/mL for group1 and 5.28 (90% CI, 3.10 – 9.48) ng/mL for group 2. The mean AMH level after first cycle of chemotherapy was 0.43 (90% CI, 0.03 – 0.81) ng/mL in all patients, 0.40 (90% CI, 0.03 – 0.69) ng/mL and 0.47 (90% CI, 0.12 – 1.09) ng/mL for each group at a mean time of 16.5 days after initial chemotherapy. The mean AMH level before local therapy was 0.17 (90% CI, 0.03 – 0.37) ng/mL and 0.07 (90% CI, 0.01 – 0.32) ng/mL at the end of chemotherapy. Conclusions: Female adolescents with Ewing sarcoma treated by VDC-IE regimen showed a rapid reduction of AMH after initial cycles. Methods for preservation of fertility should be implemented as soon as possible but not at the same time of local therapy.