The objective of this study was to determine the effects of brewer's yeast hydrolysate (BYH, Saccharomyces cerevisiae) on laying hens. A total of 288 Hy-line Brown laying hens were randomly assigned to 1 of 6 treatments with 8 replications (6 birds per replication). The treatment diets were supplemented with BYH at levels of 0, 0.05, 0.10, 0.50, 1.0 or 3.0%, respectively. The supplementation of BYH diets led to a significant linear (P < 0.05) effect on egg production, egg weight and egg mass during 37-41 and 42-46 weeks. The supplementation of BYH diets also led to a significant linear increase observed in internal egg quality: albumen height (37-41 and 42-46 weeks), Haugh unit (42-46 weeks), and yolk colour (37-41 and 42-46 weeks), and a significantly linear increase in eggshell thickness (P < 0.05) during 37-41 and 42-46 weeks. A linear increase was observed on dry matter and nitrogen at 46 weeks of laying hens, and linear increase and linear decrease were detected for Lactobacillus and E. coli at 46 weeks of laying hens, respectively. As a result, dietary BYH had beneficial effects on egglaying performance, egg quality, nutrient digestibility, and excreta microflora in laying hens.