[Objectives] The Black Stork (Ciconia nigra) is a class I protected species in China, and its diet is mainly dependent on fish resources within wetland ecosystems. Conventional dietary analysis methods, such as direct observation and gastric content analysis, are limited by challenges in sample accessibility and taxonomic resolution. This study utilized non-invasive DNA metabarcoding to examine dietary shifts between winter and summer in a C. nigra population residing in the Linzhou Qixi River National Wetland Park of Anyang City, Henan Province. [Methods] To obtain high-quality fecal samples of Black Storks for dietary analysis, we developed and implemented a rigorous sampling protocol during two seasons—winter (January) and summer (August)—of 2024. Prior to sampling, we carefully selected roosting sites of Black Storks and thoroughly cleared existing feces from these sites. On sampling days, we first excluded overlapping sites, and then collected the freshest single fecal sample from each designated roosting site. All collections were completed within the same day. A total of 20 fecal samples were obtained across both seasons, with successful DNA amplification achieved in 12 samples. After molecular confirmation of species origin, eight effective fecal DNA samples were ultimately obtained (five in winter and three in summer). DNA from these validated samples underwent amplicon sequencing using 12S rRNA markers to assess seasonal variations in dietary composition, diversity characteristics, and biomarker species. [Results] The findings revealed a trophic spectrum encompassing 2 orders, 3 families, and 14 genera. The analysis of α-diversity metrics indicated no significant seasonal variation in species richness. However, β-diversity analyses (PCoA ordination with PERMANOVA validation) revealed significant changes in dietary composition (Fig. 1), with winter assemblages exhibiting low dispersion in contrast to the increased heterogeneity observed in summer. During winter, the diet was highly specialized, with Hemiculter (Cypriniformes; 98.4% relative abundance) being dominant. In contrast, the summer diet was diverse, including Gnathopogon (22.7%, Cypriniformes), Sarcocheilichthys (19.2%, Cypriniformes), and Pseudobagrus (19.2%, Siluriformes) (Fig. 2). The analytical results from LEfSe and metagenomeSeq further supported the above findings. The specialized dietary strategy in winter might be linked to limited fish activity in cold environments, whereas the diversified dietary strategy in summer benefited from the more abundant resources and expanded habitats during the breeding season (Fig. 3). [Conclusion] The dietary composition of Black Storks exhibits significant seasonal restructuring, which is closely related to the abundance of fish resources. Conservation strategies highlight the need to protect Cypriniformes resources, develop winter emergency strategies, and preserve varied river habitats during summer to support the abundance of benthic fish spawners. This study provides evidence for adaptive management strategies for C. nigra and underscores the potential of molecular ecological tools in the preservation of endangered species.