This study was conducted to investigate the effects of selenium (Se) and vitamin E on performance and blood biochemical indices of dairy cows. Fifty-four Holstein dairy cows (60 to 100 days after calving) were selected and assigned to nine groups by unit block method based on milk yield and parity with 6 cows in each group. There were 2 stages with 8 experimental groups and 1 calibration group per stage, and the calibration group was fixed. Se and vitamin E were added according to a 4×4 two-factor design. Supplemental levels of Se were 0, 0.1, 0.2 and 0.3 mg/kg, and those of vitamin E were 0, 30, 60 and 90 IU/kg, respectively. There was 16 diets in all, and they were represented in 0/0, 0.1/0, ……, 0.2/90 and 0.3/90 groups, respectively. The pre-experimental and experimental periods lasted for 7 and 14 days, respectively. The performance and physiological biochemical indices of dairy cows were determined. The results showed as follows: 1) the milk yield in 0.3/0, 0.2/30, 0.3/30, 0.1/60, 0.2/60, 0.3/60 and 0.1/90 groups was significantly higher than that in the other groups (P<0.05); the milk fat percentage in 0/0 group was extremely significantly lower than that in 0.3/30, 0/60 and 0.3/60 groups (P<0.01), and significantly lower than that in the other groups (P<0.05); the milk total solid in 0.3/60, 0/90 and 0.2/90 groups was significantly lower than that in the other groups (P<0.05); the somatic cell count in milk in 0.3/30, 0.3/60, 0.1/90, 0.2/90 and 0.3/90 groups was significantly lower than that in 0/0, 0.1/0, 0/30 and 0/60 groups (P<0.05). 2) The serum total protein and urea nitrogen concentrations in 0.3/0 and 0.3/30 groups were significantly lower than those in the other groups (P<0.05 or P<0.01); the serum insulin and glucagon concentrations in 0.3/30 and 0.3/60 groups were extremely significantly higher than those in 0/0 group (P<0.01); the thyroxin concentration in 0.3/0, 0/30, 0.3/30, 0/60, 0.1/60, 0.2/60, 0.3/60 and 0/90 groups was extremely significantly lower than that in 0/0 group (P<0.01), and significantly lower than that in the other groups (P<0.05); the triidiothyronin concentration in 0.3/30 and 0.3/60 groups was extremely significantly higher than that in the other groups (P<0.01). 3) The serum glutamic oxalacetic transaminase activity in the groups supplemented with 0 to 0.3 mg Se and 0 to 30 IU kg vitamin E and 0/60 group was significantly lower than that in the other groups (P<0.05); the serum activities of glutamic-pyruvic transaminase in 0.3/60 group and glutathione peroxidase in 0.3/30 and 0.3/60 groups were extremely significantly lower than those in 0/0, 0.1/0, 0/30, 0/60, 0/90 and 0.3/90 groups (P<0.01). In conclusion, the optimal levels of Se and vitamin E are 0.3 mg and 30 to 60 IU/kg, respectively.