To investigate whether probiotics can regulate nonspecific immune enzyme activities, blood biochemical indexes and expression of related genes of Cynoglossus semilaevis under ammonia nitrogen stress, Bacillus subtilis Y2 as a probiotic was fed to C. semilaevis, and then ammonia nitrogen stress was applied to C. semilaevis. During the stress, Y2 was continuously fed and the related indexes were monitored. The results of macroscopical growth indexes showed that the body weight and length of the C. semilaevis in the ammonia nitrogen stress groups were lower than those of the non-ammonia nitrogen stress groups, and the figures for the Y2 groups were higher than those of the blank groups both with and without ammonia nitrogen stress. The results of immune enzyme assays showed that the activities of catalase (CAT), acid phosphatase (ACP), peroxidase (POD) and alkaline phosphatase (AKP) of C. semilaevis were increased after ammonia nitrogen stress; the activities of ACP, CAT and POD in the two groups fed with Y2 strain with and without ammonia nitrogen stress were higher than those in the control groups. The results of blood biochemical indexes showed that the contents of albumin (ALB) and globulin (GLB) in serum of C. semilaevis in the non-ammonia nitrogen stress Y2 group were slightly higher than those in the control group, the albumin/globulin ratio (A/G) was higher, and the contents of triglyceride (TG) and cholesterol (CHO) had little difference; under ammonia nitrogen stress, the contents of ALB and CHO in the serum of C. semilaevis in the control group was decreased, the content of GLB was slightly increased, the total protein (TP) and total fat in serum were decreased, and the A/G was decreased. However, Y2 could increase the content of ALB, TG and CHO, under the condition of ammonia nitrogen stress resulting in the increase of the A/G. At the same time, the content of malondialdehyde (MDA), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in serum was significantly increased under ammonia nitrogen stress. Y2 could reduce the content of MDA, AST and ALT both with and without ammonia nitrogen stress. The results of the related gene expression showed that the expression levels of heat shock protein gene (HSP70) and hemoglobin α 1 gene (HB-α1) in intestine, liver, muscle and gill of C. semilaevis were increased under ammonia nitrogen stress, and the expression of HSP70 in Y2 group was higher than that in control group, with the increase most obvious in liver tissue. On the contrary, Hb-α1expression in Y2 group decreased, especially in liver and muscle tissue. In addition, the expression of growth factor gene (IGF) in intestine, liver, muscle and gill of C. semilaevis was decreased under ammonia nitrogen stress, but the expression of IGF in both Y2 groups was higher than that in control groups both with and without ammonia nitrogen stress. In conclusion, Y2 can alleviate the effects of ammonia nitrogen stress on the immune ability, blood biochemical indexs, oxygen transport, stress ability and growth of C. semilaevis, reducing the negative effects of ammonia stress on C. semilaevis. It has a good prospect for application in the aquaculture industry.