Highland barley， as a characteristic grain in the Qinghai Tibetan region of China with more abundant active substances compared with common crops. And the future development prospects of highland barley are considerable. In this study， three kinds of highland barley powder were selected for frying and processing. The purpose of this study was to explore the effects of different kinds of highland barley powder on anti-fatigue， hypoglycemia and anti-hypoxia in mice. Compared with the control group， the weight-bearing swimming time of mice in the black highland barley fried powder group was 1.9 times higher than that in the control group， and the blue highland barley fried powder group was 1.28 times higher than that in the control group. The anti-fatigue effect of highland barley fried powder was significantly improved (P<0.05)， and different kinds of highland barley could inhibit the contents of urea nitrogen (BUN) and creatine phosphokinase (CK) in mice. The results of normal pressure hypoxia tolerance test in mice showed that different kinds of highland barley powder and highland barley fried powder had no toxic effect on heart and brain tissue of mice. The level of HIF-1 in brain tissue of mice in yellow highland barley powder， blue highland barley powder， yellow highland barley fried powder and blue highland barley fried powder group was higher than that of the control group， which was more beneficial to the improvement of hypoxia tolerance of mice. In the hypoglycemic experiment of diabetic model mice， on the one hand， highland barley powder does not produce toxic effect， on the other hand， blue highland barley fried powder has a certain effect on the improvement of biochemical indexes such as TC， MDA， GHb and so on. This study provides a scientific basis for analyzing and comparing the efficacy evaluation of different varieties of highland barley， and also provides a new idea for the development of functional food for diabetic people and potential diseased people in plateau areas.