BACKGROUND: Intensive aquaculture and commercialization production are believed to be needed for improving fish health status and probiotics are candidates to achieve this goal. OBJECTIVES: In this study, we utilized Lactobacillus plantarum and Lactobacillus pentosus as autochthonous food supplementation for biochemical investigation in rainbow trout. METHODS: In the current study, 240 juvenile rainbow trout with an average weight of 15 ± 2 g were randomly divided into four groups with three replications. Group 1, 2, and 3 were fed with 108 CFU/g of L. plantarum, L. pentosus, and the combination of these two probiotics, respectively. The control group was fed with the normal diet, for 60 days (2 % of body weight, 3times per day). Samples were taken at days 0 and 60 and biochemical parameters and hepatic enzymes activity were then investigated. RESULTS: Most biochemical factors had positive changes in the fish fed with probiotics, particularly in L. plantarum group (p < 0.05). Furthermore, the results exhibited a significant decrease in ALP level in the probiotic groups, particularly in L. pentosus group (p < 0.05). Meanwhile, AST enzyme activity in plantarum+pentosus group was higher than that in the other groups (p < 0.05). CONCLUSIONS: The present results indicated the effectiveness of food supplementation with probiotics in fish diet with the preference of probiotic to improve the biochemical factors and hepatic enzymes activity of rainbow trout, Oncorhynchus mykiss.