Abstract: Alkyl ionic liquids (AILs) are considered to be green organic solvents, but recent studies have revealed their potential ecotoxicity, which is influenced by the length of alkyl chains. The fish gut serves as a vital mechanical and immune barrier against exposure to environmental toxins, necessitating further investigation into the toxicity of different alkyl chains on the fish gut microecosystems. Therefore, in order to explore the toxic effects of three different AILs (C₆mimCl, C₈mimCl, C₁₂mimCl) on intestinal of Cyprinus carpio L., a total of 360 carps were allocated into 4 groups with 3 replicates of 30 fishes each, with the long exposure concentrations of C₆mimCl, C₈mimCl and C₁₂mimCl (the exposure concentrations were 4.97, 2.15 and 0.09 mg/L respectively) for 30d. The results are as follows: (1) As the alkyl chain length increased, the intestinal villus height and muscle thickness of carps decreased significantly (P<0.05); (2) The C₈mimCl group exhibited a significant increase in T-AOC level (P<0.05); The C₁₂mimCl group increased significantly in levels of GSH-Px, LDH, ACP and T-AOC (P<0.05); (3) The expressions of proinflammatory cytokines IL-6 and IL-8 mRNA were significantly increased (P<0.05), particularly in the C₁₂mimCl group; (4) Sequencing results showed that the microbial Chao1 and Ace indices of C₆mimCl and C₁₂mimCl groups were significantly increased (P<0.05). The results of PCoA and UPGMA cluster analysis showed that the three AILs exposures changed the intestinal flora composition of the carp. The abundance of Firmicutes which was considered as probiotic bacteria in C₈mimCl and C₁₂mimCl groups decreased significantly (P<0.05). In all the AILs exposure groups, the abundance of pathogenic bacteria such as Flavobacterium increased significantly (P<0.05), while the abundance of beneficial bacteria such as Cetobacterium decreased. In conclusion, different alkyl chains exposure can cause the damage of intestinal morphology, induce oxidative stress and immune imbalance, and change the intestinal flora structure in carp. The prediction analysis of PICRUSt2 flora function showed that the abundance of pathways related to intestinal flora metabolism in C₆mimCl and C₁₂mimCl groups increased. In total, this study revealed that the toxicity on the intestinal of carp with increasing alkyl chain length, thereby enhancing understanding of the mechanism by which AILs with different alkyl chain lengths exert toxicity on fish in aquatic ecosystem.