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The experiment aimed to investigate the effects of Bacillus subtilis and Enterococcus faecium on the production performance, egg quality, serum biochemical indexes, immune indexes, and intestinal microbes of laying hens. A total of four hundred .38-week-old Hyline Brown laying hens were randomly divided into four groups, with five replicates per group and 20 hens per replicate. Group 1 (control group) were fed with a basic diet. Group 2 received a basic diet supplemented with 200 g/t of Bacillus subtilis. Group 3 received a basic diet supplemented with 200 g/t of Enterococcus faecium. Group 4 received a basic diet supplemented with a composite probiotic consisting of 100 g/t of Bacillus subtilis and 100 g/t of Enterococcus faecium. The experimental period lasted for 56 days. The results showed that compared to the control group, the egg production rate of all the experimental groups increased (P<0.05), and the mortality rate and feed to egg ratio decreased (P<0.05). The eggshell thickness and yolk color increased (P<0.05). The total protein and globulin levels in the serum of laying hens in all the experimental groups increased (P<0.05), and the levels of immunoglobulin G, immunoglobulin M, and y -interferon in the serum all increased (P<0.05). The number of lactic acid bacteria in the cecum chyme of the laying hens increased (P<0.05), while the number of Escherichia coli and Salmonella decreased (P<0.05). The study suggests that Bacillus subtilis and Enterococcus faecium can improve the production performance of laying hens, maintain intestinal health, and enhance serum biochemical and immune indexes. [ABSTRACT FROM AUTHOR]