<p>Mechanical ventilation (MV) is a critical life-saving intervention for children with severe pneumonia-associated respiratory failure. However, prolonged ventilation significantly increases the risk of complications such as ventilator-associated pneumonia and diaphragmatic dysfunction. Therefore, there is an urgent need for biomarkers that can predict the duration of mechanical ventilation early. Mitochondrial dysfunction plays a central role in the systemic metabolic stress induced by pneumonia. Due to technical limitations in directly monitoring mitochondrial integrity at the clinical bedside, peripheral whole blood mitochondrial DNA copy number (mtDNA-CN) has been recognized as a valid surrogate marker reflecting systemic mitochondrial content. This prospective study aims to evaluate the predictive value of early-admission peripheral whole blood mtDNA-CN levels for the duration of mechanical ventilation in children with severe pneumonia. This single‑center prospective cohort study enrolled children with respiratory failure caused by pneumonia who required mechanical ventilation. Baseline clinical and laboratory data were systematically collected. Peripheral whole blood samples obtained within 24&#xa0;h of admission were used to extract total DNA. mtDNA‑CN was measured by absolute quantitative PCR targeting the mitochondrial mt‑ND1 gene. To eliminate inter‑sample variability in cell density, all copy numbers were normalized to total genomic DNA concentration and log₁₀‑transformed. Stratified analyses were performed based on the duration of mechanical ventilation (&lt; 10 days vs. ≥10 days). Statistical analyses included univariate and multivariate regression models to evaluate the association of mtDNA‑CN with ventilation duration and hospital length of stay, and ROC curve analysis was used to assess its predictive value. This prospective cohort study enrolled 72 mechanically ventilated pediatric patients admitted to the Pediatric Intensive Care Unit between June 2022 and December 2023. Patients were stratified into long-term (≥ 10 days, <i>n</i> = 33) and short-term (&lt; 10 days, <i>n</i> = 39) ventilation groups. Peripheral blood mtDNA-CN was significantly reduced in the long-term mechanical ventilation group compared to short-term ventilation patients. Univariate analysis demonstrated a substantial association between decreased mtDNA-CN and prolonged mechanical ventilation. Multivariate analysis confirmed mtDNA-CN as an independent predictor of long-term mechanical ventilation. ROC analysis revealed excellent discriminatory capacity with an area under the curve of 0.922 for mtDNA-CN. The findings of this study suggest that mtDNA copy number in peripheral blood may serve as a potential non‑invasive biomarker for detecting the duration of mechanical ventilation in children with pneumonia‑associated respiratory failure, offering advantages over other invasive markers in clinical application. However, it should be noted that more comprehensive multicenter prospective cohort studies are needed before this conclusion can be applied clinically.</p>