Intestinal microflora of grass carp (Ctenopharyngodon idellus) feeding with xylo-oligosaccharide for 56 days by concentrations of 0.1%, 0.2%, 0.4% and 0.6% respectively was investigated in this paper. Numbers of E. coli, Aeromonas and Bifidobacterium were analyzed before feeding and after 14, 28, 42, 56 days. The results showed that there was a certain influence on the intestinal microflora of grass carp after fed with different concentrations of xylo-oligosaccharides. The quantity of E. coli in the intestine of grass carps was smallest on 28th day, and compared with the control group, 0.4% group significantly different (P<0.05) has the highest decreasing magnitudes. The quantity of Aeromonas reduced but there was no significant difference with control. The quantity of Bifidobacterium increased and there was significant difference (P<0.05) between control and 0.4% group on 14th day. Therefore, feedstuffs with xylo-oligosaccharide, which were conducive to maintain a healthy state of intestinal microflora, group with 0.4% has the best effect.