This research was conducted to evaluate the effect of different levels of Alphamune prebiotic on growth performance and blood chemistry of rainbow trout (Oncorhynchus mykiss) juvenile. Rainbow trout with an initial weight of 16.46±0.09 g were randomly assigned to four dietary treatments for eight weeks. The dietary treatment was: 0 (ALP0), 1 (ALP1), 1.5 (ALP1.5), and 2 (ALP2) g of Alphamune prebiotic/kg of basal diet. The results showed that there was no significant difference in survival rate and SGR at the end of the trial. A significant difference was observed in the final weight and weight gain, and the highest one was obtained in ALP1.5 treatment (p<0.05). Food conversion rate (FCR) was affected by Alphamune levels and the lowest FCR was observed in ALP1.5 treatment. Blood biochemistry assay revealed that glucose, triglyceride, and cholesterol were not influenced by Alphamune's different levels. There were significant differences in IgM and total protein and the highest value was obtained in ALP2. These results showed that 1.5% Alphamune prebiotic (ALP1.5) had a positive effect on growth performance and biochemical parameters of rainbow trout juveniles.