The present work was conducted to evaluate the effect of dietary two levels of vitamin E (250 and 500 mg/Kg diet) with/or without single dose of thyroid hormone Thyroxin (0.25 mg/Kg diet), on the biochemical and physiological markers of Laying hens (ISA Brown Strain).  A total of  120, 29 weeks-old layer, were housed in 120 pens and assigned to 6 diets groups, first group  (control, receiving basal diet), the second and third groups received basal diet supplemented with vit-E 250 and 500 mg/kg diet respectively, the forth group kept on the basal diet supplemented with a single level of thyroid  hormone 0.25mg/kg diet; the fifth and sixth groups received basal diet supplemented with the same level of thyroid hormone combined with the two levels of vit-E/kg diet. The study was accomplished and still for 8 weeks. Finally, dietary vit-E in second level and thyroid hormone improved physiological and antioxidant parameters and decreased lipid peroxidation as confirmed from lowering in malonic dialdehyde (MDA) and enhanced GPx activity, B and Gamma globulin. Moreover, it increased HDL-C, decreased triglycerides, LDL-c and risk ratio TC/HDL-C in laying hens (ISA Brown Strain).Combining both of thyroxine and V. E had a more pronounced effect than adding each solely in comparison to the control group.