To investigate the variation in gut microbial diversity with food composition in Coilia nasus, 16S rRNA high-throughput sequencing technology was used to analyze the gut microbiota diversity of three different body length groups (L1: ≤140 mm, L2: 141–200 mm, L3: ≥201 mm) of C. nasus in the Yangtze River estuary. The analyses showed that copepods and nematodes were the main baiting taxa for C. nasus of the different length groups, and the clustering results showed that L1 was clustered into a single group, while L2 and L3 were clustered into a single group. A total of 726 genera and 378 families of microorganisms belonging to 30 phyla were identified in the intestinal samples. At the phylum classification level, the dominant groups of microorganisms in the different groups of C. nasus were similar, and were mainly composed of Actinobacteriota and Proteobacteria. Planctomycetota was only found in the intestines of L3 group. Rhodococcus was the common dominant genus in the intestines of C. nasus of different body lengths, with the highest abundance in group L1 (82.0%) and the lowest in group L3 (27.0%). Photobacterium was present at a certain percentage in all three groups. Alpha diversity analysis revealed that the gut microbial diversity of C. nasus increased with body length. Clustering analysis using the Bray-Curtis distance algorithm revealed that the clustering results of gut microorganisms among different groups were consistent with the clustering results of the stomach contents analysis. L1 was clustered into one group alone, and L2 and L3 were clustered into one group, which indicated that the composition of the food was closely related to that of the intestinal flora. Principal coordinates analysis showed that the L1 samples were separated from those of the other two groups, while the L2 and L3 samples were clustered together, which indicated that there was a difference in intestinal flora between the three groups. This study revealed that the food composition, feeding habits, and living environment of C. nasus were closely related to the composition of the intestinal microflora, which may provide a scientific basis for the conservation and rational development of C. nasus resources.