A total of 448 50-week-old Hy-Line Brown laying hens were randomly divided into 4 treatment groups with 7 replicates per group and 16 hens per replicate. Laying hens in the control group were fed a basal diet, and laying hens in the experimental groups were fed the basal diet supplemented with 0.15, 0.30 and 0.45 mg/kg selenium yeast(SY), respectively. The pre-test period was 10 days, and the formal period was 56 days. At the end of the experiment, the antioxidant, immune performance and intestinal microbiota structure of laying hens were detected. The results showed as follows: 1) dietary SY supplementation significantly increased serum immunoglobulins(IgA, IgM, IgG) levels, and significantly decreased the contents of interleukin(IL-1β, IL-2, IL-6) and tumor necrosis factor(TNF-α); 2) dietary supplementation of 0.15 and 0.30 mg/kg SY significantly increased the activities of glutathione peroxidase and superoxide dismutase in jejunum; 3) dietary SY supplementation significantly increased the relative mRNA expression of Toll-like receptor 2 gene in ileum, and the relative expression of 0.30 mg/kg SY group was the highest, while dietary supplementation of 0.45 mg/kg SY significantly increased the relative mRNA expression of transforming growth factor β3 gene in jejunum; 4) dietary supplementation of 0.30 mg/kg SY significantly increased the relative mRNA expression levels of zonula occludens 1 and occludin genes in ileum and jejunum, and dietary supplementation of 0.30 and 0.45 mg/kg SY significantly increased the relative mRNA expression levels of zonula occludens 2, claudin 1 and mucin 2 genes in jejunum; 5) the dominant bacteria in the cecum of laying hens in each group were Firmicutes and Bacteroidetes, the dominant bacteria were Rikenellaceae_RC9_gut _group and Bacteroides, and dietary supplementation with 0.45 mg/kg SY significantly increased the relative abundance of Desulfobacterota and Desulfovibrio. In summary, the addition of SY in the feed can improve the antioxidant and immune performance of laying hens in the late peak laying period; and under the conditions of this experiment, the suitable addition amount of SY was 0.30 mg/kg.