The present study was conducted to determine the optimal supplementation level of beta-mannanase in the diet of laying hens. A total of 320 Hy-Line Brown layers (80 weeks of age) were assigned randomly into four groups on the basis of laying performance. Each treatment had eight replicates with 10 birds each (80 birds per treatment). Two hens were caged individually. Treatments were basal diet supplemented with 0 (control), 0.04, 0.08, and 0.16% beta-mannanase during the nine-week feeding period. Laying hens fed diets supplemented with increasing levels of beta-mannanase had increased (linear, p<0.05) overall egg production and egg mass. In addition, these hens had greater retention of dry matter, crude protein, gross energy, calcium, and mannan (linear, p<0.05). Dietary beta-mannanase treatments had no effect on blood metabolites such as total carbohydrate, triglycerides, glucose, total protein, and blood urea nitrogen, or excreted ammonia nitrogen and volatile fatty acids. The results obtained in present study indicate that dietary supplementation of beta-mannanase has the potential for improving the performance of laying hens. The optimal supplementation level is 0.04% beta-mannanase in the diet.