Fish occupy pivotal trophic positions in aquatic ecosystems, mediating energy transfer and shaping community structure through their feeding interactions. Unraveling these dietary relationships is therefore fundamental for understanding ecosystem functioning and supporting sustainable fisheries management. Traditional morphological analyses, while informative, often fall short in resolving fine-scale prey diversity and trophic linkages. In contrast, DNA metabarcoding has revolutionized dietary studies by enabling comprehensive, high-resolution, and non-invasive characterization of prey assemblages. This review synthesizes recent progress in applying DNA metabarcoding to fish trophic ecology, emphasizing technical innovations, methodological standardization, and ecological insights. We discuss how DNA metabarcoding has advanced the understanding of food web complexity, species interactions, and ecological responses to environmental change. However, challenges persist in quantification accuracy, reference database completeness, and cross-source contamination. Future research integrating multi-marker approaches, standardized workflows, and multi-method integration holds promise for transforming DNA metabarcoding into a powerful, reliable and mechanistic tool for trophic ecology. Collectively, these developments will bridge molecular data with ecological theory, strengthening the scientific foundation for ecosystem-based fisheries management.