The study was conducted to test the effects of dietary probiotics (Clostridium butyricum, Bacillus coagulans) on biochemistry, non-specific immunity and disease resistance in the serum of Rainbow Trout, Oncorhynchus mykiss. The study produced the following results. First, compared to the control treatment T0, there was a higher increase of the contents of total protein and glucose in the serum of O. mykiss fed with signal and multiple probiotics (P0.05), and meanwhile there was a decrease of the contents of total-cholesterol and triglycerides (P0.05). Second, the combination of multiple probiotics, Clostridium butyricum and Bacillus coagulans, was thought to be even more beneficial than a single probiotic. The activities of acid phosphatase (ACP), total antioxidant capacity (T-AOC), catalase (CAT), superoxide dismutase (SOD) in treatment T3 were significantly improved (P0.05), and the content of malondialdehyde (MDA) was significantly reduced, with treatment T2 with Bacillus coagulans having a better effect than treatment T1 with Clostridium butyricum. The content of Complement C3 in treatment T3 with mixed probiotics was significantly increased. These could enhance the non-specific immunity of O. mykiss. Third, supplementation of different probiotics could decrease the cumulative mortality of O. mykiss and improve the relative percent survival (RPS). The lowest cumulative mortality and the highest RPS of O. mykiss were found in treatment T3, they were 23.1% and 66.6% respectively. It can be concluded that the supplementation of Clostridium butyricum and Bacillus coagulans in diets can enhance the immunity of O. mykiss.