A 4-week feeding trail was conducted to explore the impacts of dietary binder sources on growth performance, digestive enzymes activities, intestinal microbiota, and expression of genes involved in tight junctions and nutrient sensing pathway of larval largemouth bass with initial body weight of 8.43 ± 0.01 mg (13 day after hatching). Five isolipidic micro-diets were formulated with different binder sources including sodium alginate (SA), carrageenan (CG), hydroxypropyl methyl cellulose (HPMC), gelatin (GT), and starch (SC). The results revealed that the highest growth performance was observed in the CG group, and meanwhile, the promotion effects of SA and GT was statistically higher than that of HPMC and SC groups. SA, CG and GT had positive effects on digestive enzymes activity compared to other groups. Gene expression results showed that SA, CG and GT groups up-regulated the expression of tight junction genes, zo-1, claudin-4 and claudin-7, and activated the genes involved in the TOR signaling pathway, including tor, akt1, s6kβ1 and s6. Among all the groups, the CG had the most significant activation effect on TOR signaling pathway. Bacterial 16S rRNA V3–4 region analysis showed that the addition of different dietary binders influenced the diversity of intestinal microbial communities. The inclusion of SA and CG groups reduced the relative abundance of Proteobacteria and Actinobacteria at the phylum level, while at the genus level, the relative abundance of Rhodococcus and Achromobacter increased and the abundance of Plesiomonas was inhibited. In summary, dietary binder sources affected the growth, digestive function, gut barrier function and microbial composition, and nutrient sensing, and SA and CG could be suggested as the optimal dietary binders for formulation of micro-diets for largemouth bass larvae.