This study investigates the protective effects of Lactiplantibacillus plantarum Lp05 on ethanol-induced liver injury in zebrafish (Danio rerio), focusing on its mechanisms related to metabolic pathway regulation and gut microbiota composition. We administered Lp05 to zebrafish exposed to ethanol to assess its impact on liver enzymes and overall liver health. Key metrics included the expression levels of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH), activity of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and changes in gut microbiota composition through 16S rRNA sequencing. Metabolomic analyses were conducted to identify affected metabolic pathways. Treatment with Lp05 significantly increased ADH and ALDH expression, enhancing alcohol metabolism and reducing ALT and AST activities, thereby mitigating liver cell damage. Metabolomic analysis revealed significant modulation of biotin and glycerolipid metabolism pathways, crucial for reducing liver injury. Lp05 also altered gut microbiota, increasing beneficial bacteria such as Fluviicola and Delftia, while decreasing pathogenic bacteria like Acinetobacter and Aeromonas. This bacterial modulation contributed to phenylalanine metabolism regulation, which alleviated intestinal inflammation and liver injury. Additionally, Lp05 promoted the degradation of polycyclic aromatic hydrocarbons (PAHs), reducing their health risks. Lp05 exhibits potential therapeutic effects against ethanol-induced liver injury in zebrafish by enhancing alcohol metabolism, modulating metabolic pathways, and altering gut microbiota. These findings suggest that Lp05 may offer a novel preventative and therapeutic strategy for managing alcoholic liver injury. Alcohol-induced liver injury is a significant global health concern with limited effective therapeutic options. This study highlights the potential of Lactiplantibacillus plantarum Lp05 as a novel probiotic intervention for mitigating ethanol-induced liver damage. By enhancing alcohol metabolism, regulating critical metabolic pathways such as biotin and glycerolipid metabolism, and modulating gut microbiota composition, Lp05 addresses both the metabolic and microbiological aspects of liver health. The promotion of beneficial bacteria and the suppression of pathogenic strains further contribute to alleviating liver injury and systemic inflammation. These findings underscore the therapeutic promise of Lp05 in managing alcoholic liver injury and provide a foundation for its development as a preventative and therapeutic strategy in human applications.