Objective: To investigate the effects of midazolam (MDZ) combined with fentanyl on stress response and hemodynamics in patients undergoing laparoscopic ovarian cyst resection (LOCR). Methods: The clinical data of 94 patients treated with LOCR in our hospital from August 2022 to June 2024 were retrospectively collected. Patients were divided into control group (receiving fentanyl anesthesia, 47 cases) and observation group (receiving MDZ combined fentanyl anesthesia, 47 cases) according to different anesthesia protocols. The hemodynamic level [mean arterial pressure (MAP), heart rate (HR)], pain degree [visual analogue scale (VAS) score], sedation effect, stress response [glutathione peroxidase (GSH-Px), total antioxidant state (TAS), hydrogen peroxide (H2O2)] and complication rate of the two groups were analyzed and compared. Results: The levels of MAP and HR after sedation and immediately after recovery were lower than those before medication, but the levels of MAP and HR in the observation group were significantly higher than those in the control group (P < 0.05). The VAS scores of the observation group were significantly lower than those of the control group at 6h, 12h and 24h after operation (P < 0.05). The good and good rate of sedation in observation group was significantly higher than that in control group (P < 0.05). Compared with before surgery, serum GSH-Px and TAS levels were decreased and H2O2 levels were increased in both groups 12h after surgery, but serum GSH-Px and TAS levels in observation group were significantly increased compared with control group, and H2O2 level was significantly decreased compared with control group (P < 0.05). There was no significant difference in the incidence of complications between the two groups (P > 0.05). Conclusion: MDZ combined with fentanyl in patients with LOCR can improve sedation, relieve pain, reduce the impact on the body"s hemodynamics and stress response, with high safety.