High-concentration oxygen inhalation is the primary intervention to prevent perioperative hypoxemia. However, there are concerns that this may induce an imbalance in oxidation‒reduction processes, particularly in pediatric patients with compromised antioxidant defenses. This study aimed to evaluate the impact of varying intraoperative concentrations of oxygen inhalation on oxidative stress in children by analyzing plasma biomarkers, oxygenation indices, and the duration of surgery and oxygen inhalation. Forty-five children scheduled for laparoscopic pyeloplasty under general anesthesia were randomly assigned to three groups, each receiving different fractions of inspired oxygen during surgery: 30%, 50%, or 80%. The primary outcome was the plasma concentration of oxidative stress markers, and the other measurements included the surgical duration and duration of oxygen exposure. Thirty-five children completed the study, with 11 in the low group, 12 in the medium group and 12 in the high group. The levels of superoxide dismutase at immediately post-tracheal intubation, hydrogen peroxide at 1 hour post-intubation, and 8-isoprostane at immediately post-surgical procedure were significantly higher in the high group than in the low group. The S100B levels at immediately post-surgical procedure were higher than those at immediately post-tracheal intubation and 1 hour post-intubation within the low group. Therefore, we conclude that inhaling a high concentration of oxygen during laparoscopic pyeloplasty under general anesthesia, for a duration of less than 3 hours, does not significantly increase oxidative stress in pediatric patients. This study was registered at the Chinese Clinical Trial Registry (registration No. ChiCTR2400083143).