Abstract: The effects of bio-fertilizers on the growth of highland barley (Hordeum vulgare) in alpine regions were studied by using the split-block design experiment. The bio-fertilizers were composited with the associative nitrogen-fixing bacteria (Pseudomonas sp., Azotobacter sp., Zoogloea sp.). The results indicate that the associative nitrogen-fixing bio-fertilizers accord with the quality criterion of agricultural microbial fertilizer of China (GB 20287-2006). The root length (dough stage), underground plant biomass (mature stage), crude protein (heading stage, postulation stage and dough stage) and grain yield are significantly promoted by the bio-fertilizer applications (P0.05). In the 1/2CN treatment group (bio-fertilizer + half application rate of chemical nitrogen fertilizer), the height (seedling stage), aboveground biomass (postulation stage, dough stage and mature stage), root length (dough stage), root volume (postulation stage), underground biomass (dough stage and mature stage), crude protein (whole stage) and grain yield are significantly higher than those of the control (P0.05). The plant height (heading stage, postulation stage and dough stage), chlorophyll content (the whole stage), aboveground biomass (heading stage and dough stage), root length (postulation stage and mature stage), root volume (heading stage, postulation stage and mature stage), underground biomass (the whole stage), crude protein (mature stage) of the 1/2CN treatment are lower than those of the CN treatment (the whole application rate of chemical nitrogen fertilizer), and the differences are not significant. The 1/2CN treatment shows the similar results with the CN treatment in certain growth stages of highland barley, therefore, the 1/2CN treatment could save half amount of chemical nitrogen fertilizer (56.25 kg/ha) in this region.