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Pediatric acute liver failure (PALF) is a severe liver dysfunction with complex pathological mechanisms and rapid development. MiRNAs have been identified as promising biomarkers for human disease screening and monitoring. This study focused on evaluating the clinical significance of miR-224-5p in PALF and revealing its potential molecular mechanism in regulating liver cell injury. This study enrolled 103 children with PALF and 55 healthy children without liver diseases. Serum miR-224-5p levels were compared between the two groups, and their clinical significance was estimated by analyzing the correlation with clinicopathological features and outcomes of PALF children. In vitro, a normal liver cell was treated with lipopolysaccharide (LPS), and cell growth and inflammation were assessed by CCK8 and ELISA assay. Upregulated miR-224-5p in PALF showed significance in screening PALF children from healthy children with the sensitivity and specificity of 78.64% and 84.47%, respectively. Increasing serum miR-224-5p in PALF children was closely associated with increasing prothrombin time, alanine transaminase, international normalized ratio, total bilirubin, ammonia, and aspartic transaminase and decreasing albumin of PALF children. MiR-224-5p was also identified as a risk factor for adverse outcomes in children with PALF. In LPS-treated liver cells, miR-224-5p could negatively regulate ZBTB20, and silencing miR-224-5p could alleviate the inhibited cell growth and promoted inflammation by LPS, which was reversed by ZBTB20 knockdown. Increasing miR-224-5p distinguished PALF children, predict severe disease development and risk of adverse prognosis. miR-224-5p also reguled LPS-induced liver cell injury via negatively regulating ZBTB20.