The purpose of this study was to investigate into the effects of Se-chitosan and Na selenite supplementation on laying hen production performance, egg quality, egg Se concentration, microbial population, immunological response, antioxidant enzymes activity, and yolk fatty acid profile. Using a 2 × 2 factorial design, 168 27-wk-old laying hens were randomly divided into 4 treatment groups and 7 replications. Se source (Na selenite and Se-chitosan) and Se level (0.3 and 0.6 mg/kg) were used as treatments. Se-chitosan enhanced egg production percentage and egg mass (P < 0.05) when compared with Na selenite. There was an interaction, with 0.6 mg Se-chitosan/kg causing an increase in albumen height, Haugh unit, albumen index, and shell thickness of fresh eggs (P < 0.05). Se-chitosan increased yolk share, yolk color, and shape index of fresh eggs and shape index, albumen index, albumen height, Haugh unit, yolk color, shell thickness, and specific gravity of stored eggs (P < 0.05). The interaction showed that, 0.6 mg Se-chitosan/kg increased albumen Se concentration and decreased the level of malondialdehyde (MDA) in fresh egg yolk compared with 0.3 and 0.6 mg Na selenite/kg (P < 0.05). When compared with Na selenite, Se-chitosan increased the Se concentration in the yolk and decreased level of MDA in stored egg yolk (P < 0.01). When compared with Na selenite, Se-chitosan reduced coliforms (P < 0.01), increased lactic acid bacteria, and the lactic acid bacteria/coliform ratio (P < 0.05). Se-chitosan supplementation increased antibody response to sheep red blood cells and IgM titers and the activities of glutathione peroxidase and superoxide dismutase in plasma (P < 0.05). Furthermore, compared with Na selenite, supplementing diets with Se-chitosan decreased ∑ n-6 PUFA/∑ n-3 PUFA ratio (P < 0.01). In conclusion, Se-chitosan supplementation of laying hen feed improved production performance, egg quality, egg Se concentration, yolk lipid oxidation, microbial population, immune response, antioxidant enzymes activity, and yolk fatty acid profile, with 0.6 mg Se-chitosan/kg supplementation being optimal.