In the present study, the effects of Lactobacillus rhamnosus oral administration on blood biochemical parameters of rainbow trout (Oncorhynchus mykiss) were evaluated under the stress induced by sub-lethal toxicity of malathion. A total of 180 fish with the mean weight of 43±2.6 g were divided into four experimental groups; control, oral administration of probiotic, exposed to malathion with oral administration of probiotic and exposed to malathion with probiotic free diet. Selected dose of malathion was 0.036 mg/L and the probiotic were also used at 1 g (10¹⁰ cfu/g) per kg of diet. At the end of the 28-day experiment period, the percentage of weight gain and some of the blood biochemical parameters of the fish were examined. The results showed a decrease in the percentage of weight gain in fish treated with malathion and oral administration of L. rhamnosus supplement did not have a significant effect on increase of this index (P<0.05). Providing probiotic diet to fish treated with malathion significantly increased the total protein and albumin levels compared to basal diet (P>0.05). Blood cortisol level in fish treated with malathion was higher than control group and oral administration of L. rhamnosus significantly decreased it compared to control diet (P<0.05). Plasma enzyme activity assay showed that using L. rhamnosus probiotic, except ALP of plasma did not have a significant effect on the activity of other enzymes in fish treated with malathion (P<0.05). According to the results, it can be concluded that oral administration of L. rhamnosus probiotic for rainbow trout at the time of exposure to sub-lethal toxicity of malathion does not have an effect on the reduction of tissue disorders and tissue damage but it can be useful in reducing stress and improving the immune system of the fish.