Objectives The effects of different levels of Saccharomyces cerevisiae culture on the production performance of laying hens were studied to provide reference for the utilization of diets with low soybean meal and low protein in livestock and poultry production. Methods 1 080 laying hens aged 36 weeks with similar body weight were randomly divided into 5 groups, with 6 replicates in each group and 36 chickens in each replicate. They were fed with basic diets supplemented with 0%, 2.5%, 5%, 7.5%, and 10% Saccharomyces cerevisiae, respectively. The basic diet was corn-soybean meal, and the diets of each group were isocaloric and isonitrogenic. The experiments lasted 35 days. Results The weight of egg decreased linearly, while the ratio of feed to egg, and a^* value and b^* value of eggshell color increased linearly with the increase of the level of adding Saccharomyces cerevisiae culture to the diet of laying hens. There was a quadratic change in the height of concentrated protein and the Ha unit of eggs, while the activities of alanine aminotransferase and aspartate aminotransferase in serum decreased linearly. The addition of 2.5% Saccharomyces cerevisiae culture to the diet of laying hens significantly increased the height of concentrated protein and Hasse units of eggs, while reduced the yolk color of eggs (P<0.05) compared with the control group. Diets supplemented with 7.5% and 10% Saccharomyces cerevisiae culture significantly reduced the egg quality(P<0.05). Adding 10% Saccharomyces cerevisiae culture to the diet significantly reduced the activity of aspartate aminotransferase in serum of laying hens (P<0.05). Conclusions It is indicated that adding 2.5% Saccharomyces cerevisiae culture to the diet of laying hens is the optimal dosage under the conditions of this experiment.