This experiment was conducted to study the effects of nano-selenium on performance, serum immune and biochemical indices and yolk selenium content of laying hens. A total of 324 laying hens (29 weeks of age) were randomly allotted to 6 groups with 6 replicates in each group, and each replicate contained 9 hens. Hens in the control group (group A) were fed a basal diet, and hens in experimental groups (groups B, C, D, E and F) were fed the diets supplemented with 0.1, 0.2, 0.3, 0.4 and 0.5 mg/kg nano-selenium, respectively. The adjustment period lasted for 7 days, and the experimental period lasted for 63 days. The results showed as follows:1) compared with the control group, the feed to egg ratio at the 21st day of group C was significantly decreased (P<0.05), the grain-hulling rate at the 42nd and 63rd day of groups B and D was significantly decreased (P<0.05). There were no significant effects on laying rate and average egg weight among all groups (P>0.05). 2) Compared with the control group, the contents of immunoglobulin M (IgM) and immunoglobulin A (IgA) in serum at the 42nd day of group E were significantly increased (P<0.05), and there was no significant effect on serum immunoglobulin G (IgG) content at the 42nd day among all groups (P>0.05). There were no significant effects on serum IgM, IgA and IgG contents at the 63rd day among all groups (P>0.05). 3) Compared with the control group, at the 42nd day, the serum albumin content of group D was significantly increased (P<0.05), the serum triglyceride (TG) and glucose (Glu) contents of group D were significantly decreased (P<0.01), the serum total protein (TP) content of group F was significantly increased (P<0.05), the serum uric acid (UA) content of groups E and F was significantly decreased (P<0.05). Compared with the control group, at the 63rd day, the serum TP content of group F was significantly increased (P<0.05), the serum alkaline phosphatase (ALP) activity and Glu content of group F were significantly decreased (P<0.05). 4) Compared with the control group, the yolk selenium content at the 21st day of groups C, D, E and F was significantly increased (P < 0.05 or P < 0.01), the yolk selenium content at the 42nd days of experimental groups was significantly increased (P < 0.05 or P < 0.01), the yolk selenium content at the 63rd day of groups C, D, E and F was significantly increased (P<0.01). The yolk selenium content of group F was the highest at all experiment period, and the yolk selenium content of experimental groups at the 42nd days was higher than that at the 21st and 63rd day. There were no significant effects on yolk iron, manganese, zinc and copper contents among all groups (P>0.05), except the yolk zinc content at the 42nd day of experimental groups was significantly higher than that of control group (P<0.05). In conclusion, dietary nano-selenium can improve the performance of laying hens, improve the serum immunity, promote the synthesis of proteins of organism, reduce the content of cholesterol in blood, promote the health of hens and increase the yolk selenium content. Comprehensive consideration, the optimum level of nano-selenium in the diet is 0.4 mg/kg, and the effect is more obvious when experiment last for 42 days.