Abstract: This experiment was conducted to achieve the optimal dietary fish oil supplementation level of juvenile American eels (Anguilla rostrata) by evaluating the parameters of growth performance, digestive enzymes activities, body composition, and lipid metabolism enzymes in the liver. 800 juvenile American eels with an average body weight of (8.34±0.12) g per fish were randomly divided into 4 treatment groups with 5 replicates per group and 40 fish per replicate. The basal diets were supplemented with 0 (control group), 3% (FO3 group), 6% (FO6 group), 9% (FO9 group), and 12% (FO12 group) fish oil, respectively. The trial lasted for 56 days. Dietary fish oil supplementation significantly affected the growth performance of juvenile American eels. The weight gain rate, feeding rate, and feed efficiency of juvenile American eels in FO6 group were significantly higher than those in other groups (P<0.05). Compared with the control group, intestinal lipase activities increased significantly in fish oil supplementation groups (P<0.05). The activities of protease and amylase decreased significantly by fish oil supplementation (P<0.05), and there was no significant difference in the activities of protease and amylase among different fish oil supplementation groups (P>0.05). Compared with the control group, the contents of crude lipid of the whole body increased significantly in FO6 group, FO9 group, and FO12 group (P<0.05). The content of crude protein decreased significantly only in FO12 group (P<0.05). The contents of moisture and ash were similar among all the fish oil supplementation groups (P>0.05). The activities of fatty acid synthase decreased significantly only in FO9 and FO12 groups (P<0.05), the activities of lipoprotein esterase and liver lipase increased significantly only in FO12 group (P<0.05). In conclusion, the adequate supplementation level of fish oil in diet might improve the growth performance by regulating the intestinal digestive enzymes activities, lipid content in whole fish and lipid metabolism enzymes levels or activities in the liver. The recommended level of fish oil supplementation in the diet was 6.43% to 6.78% for the highest weight gain rate and feed efficiency of juvenile American eels.