Objective To determine the 90% effective dose (ED90) of oral midazolam combined with intranasal esketamine for preoperative sedation in children and observe its feasibility. Methods A prospective double‑blind sequential study was conducted using 40 children who were scheduled for surgery according to the sequential method. The main outcomes were the oral dose of midazolam that achieved the Parental Separation Anxiety Score (PSAS) of children=1 30 min after used in combination with intranasal esketamine at a low dose. The secondary outcomes were preoperative systolic blood pressure (SBP), heart rate, sedation level, onset time, preoperative side effects, PSAS scores after administration for 30 min, the Mask Acceptance Scale (MAS) score, the reaction to intravenous cannulation scale (ICS) score, postoperative extubation time, consciousness recovery time, the length of post‑anesthesia care unit (PACU) stay, the use of additional analgesic drugs, and the incidence of adverse events such as hypoxemia, nausea and vomiting. The isotonic regression was used to analyze ED90 and its 95% confidence interval (CI). Results The ED90 of oral midazolam that used in combination with 0.25 mg/kg intranasal esketamine for preoperative sedation in children was 0.253 (95% CI 0.242‒0.278) mg/kg. The average onset time was (8.9±3.8) min. The incidence of hallucinations and excited behavior changes was 7.5%. No children had adverse reactions such as hypoxemia, hypotension, bradycardia, hypertension, tachycardia, nausea and vomiting. Conclusions The combined use of 0.25 mg/kg intranasal esmketamine and oral midazolam can effectively relieve preoperative anxiety in children, and the ED90 of midazolam is 0.253 mg/kg.