In order to explain the alpine meadow plant community characteristics and the response of soil nutrient and soil microbial activity to fertilizer gradients in Kobresia humilis meadow. Effects of different fertilizer gradients on the structure, function of plant community, contents of total soil nutrients and available nutrients, soil organic carbon and microbial biomass carbon in the K. humilis meadow were studied in Three-river Headwater Areas of Qinghai-Tibetan Plateau, China. The results showed that. 1) With the increase of fertilizer application, the coverage in different functional groups response differently. Among which the coverage of grasses function group increased a lot, while legumes and forbs function group coverage decreased obviously, there were no obvious variation in the coverage of sedges function group; however, the coverage of each plant function group will be reduce gradually when fertilizer application increased to a certain degree, for example, when N fertilizer application reached 40 g/m². Plant function group biomass showed unimodal curve changes with different fertilizer gradients and the biomass reached the minimum under unfertilizing conditions and the maximum when 20 g/m² or 32 g/m². 2) The contents of total soil nutrients and available nutrients are higher when fertilizer application is 20 g/m² or 32 g/m² and soil resource will be reduced gradually when soil application increase to 40 g/m². 3) Soil organic carbon and microbial biomass carbon of different fertilizer gradients in the K. humilis meadow were obviously higher at 0-10 cm soil layer, and the contents of the soil organic carbon at 0-40 cm soil layer showed unimodal curve variation with increasing N fertilizer application. The highest value of soil organic carbon and microbial biomass carbon were occurred in 20 g/m²or 32 g/m² conditions. 4) Fertilizer application with 30 g/m² can be the best nitrogen level to alpine meadow. The contents of soil organic carbon and microbial biomass carbon under fertilizer gradients can be an important indicator to evaluate soil fertility and soil quality variation. The content N fertilizer application (≥40 g/m²) which is used as a threshold to influence ecosystem structure, function, soil nutrient, and soil microbial activities in alpine meadow.