The intestine of fish plays an important role in carbohydrate, protein, lipid metabolism and immunity. To obtain molecular biological information to the studies of some important physiological procession in grass carp, Ctenopharyngodon idellus. A subtractive cDNA library was constructed from the intestinal tract of grass carp experimentally infected with Aeromonas hydrophila by suppression subtractive hybridization (SSH). A total of 211 cDNA clones were partially sequenced and 147 ESTs obtained from the library. BLAST result showed that 147 of the intestinal ESTs could be ascribed to the transcriptional products of 97 identified genes, whereas 50 ESTs exhibited relatively low homology with unidentified sequences. Of the 97 known ESTs，There are 43 EST of cell defense-related, belonging to 13 genes. The successful constructed Subtractive cDNA library will be essential for rapid isolation of immune-related genes of grass carp intestinal tissue and further to explore mucosal anti-infectious molecular mechanisms in fish.