Abstract: Background and Objective: Chitosan is a natural alkaline polysaccharide and widespread in nature. A study was conducted to evaluate the effect of dietary supplementation of chitosan on blood biochemical parameters like cholesterol, malondialdehyde (MDA), creatinine and total leucocytes. Methodology: One hundred laying phase hens, aged 28 weeks were used in the study. The birds were divided into 2 treatment groups each having 3 replicates of 10 birds each. Group I served as control and was fed basal diet. In Group II, basal diet was supplemented with chitosan at 150 ppm g–1 by spraying method. Results: The results showed that the chitosan inclusion in the diet of layer pullets significantly (p<0.05) lowered the total cholesterol (36.749±0.381 mg dL–1) when compared to the control group (43.030±0.352 mg dL–1). The dietary incorporation of chitosan significantly (p<0.05) decreased blood MDA levels (1.829±0.237 nmoles mL–1) of laying hens compared to control (2.553±0.379 nmoles mL–1). The creatinine levels also decreased significantly (p<0.05) in birds fed diet supplemented with chitosan, however, there was no effect on the total leucocyte count. Conclusion: The incorporation of chitosan in the diet had positive effect in terms of reducing the blood cholesterol and malondialdehyde levels of laying hens.