Background Early detection of metastasis and recurrence of Ewing sarcoma (ES) is important for early management. This work aimed to detect CD99+, CD45− cells in peripheral blood by flow cytometry (FC) before and during chemotherapy and evaluate their prognostic significance. Procedure This prospective cohort study was carried out on 60 children newly diagnosed with ES at Children Cancer Hospital‐Egypt 57357 and 40 healthy children control group. Detection of CD99+, CD45− cells in peripheral blood was accomplished by FC at baseline before treatment and after five cycles of chemotherapy. Samples were classified as positive if they had more than the upper limit of cells observed in the control cases. Correlation between FC results and relapse and overall survival (OS) after one year was performed. Results Median percentage of CD99+, CD45− cells was significantly increased in patients compared with controls (0.002% vs 0%, respectively, P < 0.001). Post‐cycle 5 CD99+, CD45− cells were increased in 12 patients, of them 11 patients’ disease had either relapsed or progressed. Post‐cycle 5 CD99+; CD45− cells had a 73.3% sensitivity and 97.8% specificity for predicting relapse or progression, whereas baseline only had 6.7% sensitivity and 77.8% specificity. The hazard ratio for mortality in the post‐cycle 5 positive group was 18.4 [95% confidence interval (1.86 to 181.46)] times that of the negative group. One year OS was 91.67%. Conclusion Post‐cycle 5 CD99+, CD45− cells in peripheral blood by FC is a strong predictor for relapse, progression, and mortality whereas baseline is a poor predictor in newly diagnosed patients with ES.