This study was conducted to evaluate the effects of different molecular masses of chito-oligosaccharide (COS) on performance, egg quality, serum biochemical indices, intestinal microorganism, IL-2 and TNF-α gene expressions in spleen of laying hens. A total of 480 seventy-week-old laying hens with similar body weight and laying rate were randomly allocated to four groups with four replicate per group and 30 birds per replicate. The laying hens in control group were fed a basal diet, and those in the test groups were fed the diets with 200 mg/kg COS that molecular mass was 1, 3 and 8 ku, respectively. The experiment lasted for 42 days. The results showed as follows: 1) no significant differences were found in feed/egg ratio, laying rate, average daily feed intake (ADFI) and average egg weight (P>0.05). 2) Compared with the control group, the supplementation of 1, 3 and 8 ku COS improved Haugh unit by 3.70% (P>0.05), 5.20% (P>0.05) and 12.71% (P<0.05) at 21 days of the experiment, respectively. 3) The contents of triglyceride and glucose in serum of laying hens of 3 ku COS group were decreased by 39.91% (P<0.05) and 5.86% (P>0.05). 4) Compared with the control group, the number of intestinal E. coli and Bifidobacteria of laying hens was decreased by the supplementation of 1 ku COS (P<0.05). 5) The TNF-α gene mRNA expression level in spleen of laying hens of 3 ku COS group was significantly higher than that of the control group (P<0.05). The supplementation of 1 and 3 ku COS significantly increased IL-2 gene mRNA expression level in spleen of laying hens (P<0.05). In conclusion, the supplementation of 3 ku COS can decrease triglyceride content in serum, improve IL-2 and TNF-α gene mRNA expression levels in spleen, enhance the immune function of laying hens, and the 3 ku COS is suggested to be added.