Animal digestive tract is habitat for a large number of autochthonous microbiota, which play central roles in multiple biological and physiological processes of the host. In this study, two different micro-biomass preparation methods were employed to evaluate the diversity of intestinal mucosa-associated microbiota in grass carp (Ctenopharyngodon idellus). Genomic DNAs were isolated either directly from intestinal mucosal samples (group A), or from micro-biomass after microbial dissociation (group B). Community richness, diversity and evenness indices were all higher in group B, but differences were not statistically significant (P=0.97, P=0.33, P=0.34 respectively). Furthermore, group B samples exhibited an increased ratio of bacterial DNA in comparison with group A samples, but the difference was also not statistically significant (P=0.74). In addition, there were no statistically significant differences between the two groups (P>0.05) at the taxonomic level. Our results support previous findings that there exists a great abundance of the intestinal mucosa-adherent microbiota in the grass carp; among these, Proteobacteria, Firmicutes, Bacteroidetes, Actinobacteria, Spirochaetes and Fusobacteria were the most common phyla. Within these microbiota, Paenibacillus, Bacteroides, Bacillus and Cetobacterium genera comprise the majority of the community, implicating their functional importance (e.g. as probiotics) to their host. Our results contribute towards a better understanding of the intestinal microbial profile of grass carp. Both micro-biomass preparation techniques proved to be feasible for studying mucosa-adherent microbiota of grass carp; however, the second method (group B) provides a protocol that is somewhat more effective than the first method (group A).