Little egret (Egretta garzetta) is widely distributed globally. It is an indicator species for assessing habitat environmental quality. This study collected fecal samples of little egrets using non-invasive sampling methods, and then sequenced the extracted fecal genomic DNA using 16S rRNA amplicon sequencing and metagenome sequencing methods. Multiple bioinformatics methods were used to compare and analyze the similarities or differences in the gut microbiota and food composition of little egrets under the influence of habitat environment and age factors, and to explore the relationship between food composition and gut microbiota.