To investigate the supplemental effects of Bacillus subtilis C‐3102 on the production, hatching performance, egg quality, serum antioxidant capacity and immune response of laying breeders, a total of 480 Xuefeng black‐bone (25‐week‐old) hens were randomly assigned into four treatment groups: Hens fed the basal diets with 0 (CON), 3.0 × 105 (BS‐1), 6.0 × 105 cfu/g (BS‐2) and 9.0 × 105 (BS‐3) cfu/g of B. subtilis C‐3102. As the B. subtilis C‐3102 level increased, egg weight (linear, p < 0.01; quadratic, p = 0.003), fertility (linear, p = 0.021; quadratic, p = 0.059), hatchability (linear, p = 0.038; quadratic, p = 0.119) and yolk colour (linear, p = 0.006; quadratic, p = 0.021) increased in a linear or quadratic manner. Yolk index increased quadratically (linear, p = 0.054; quadratic, p = 0.017), and eggshell thickness (linear, p = 0.036; quadratic, p = 0.128), the activity of GSH‐Px (linear, p = 0.024; quadratic, p = 0.078), the concentration of IgM (linear, p = 0.016; quadratic, p = 0.056) and the level of AIV‐Ab (linear, p = 0.034; quadratic, p = 0.103) in the serum increased linearly as dietary supplementation of B. subtilis C‐3102 increased. The results showed that dietary treatments did not affect egg production, feed conversion ratio, egg mass, hatchability of fertile eggs, eggshell‐breaking strength, egg‐shape index, yolk percentage, Haugh unit, T‐SOD, T‐AOC, MDA, IgA and IgG concentrations and the level of NDV‐Ab in the serum. In conclusion, dietary supplementation of 9.0 × 105 cfu/g B. subtilis C‐3102 in laying breeders diets may be a feasible means of effectively increasing egg weight, fertility and hatchability, and improving egg quality such as eggshell thickness, yolk index and yolk colour. Besides, B. subtilis C‐3102 can enhance the activity of GSH‐Px, the concentration of IgM and the level of AIV‐Ab in the serum.