In order to reveal the spatial and temporal variations of soil microbes in alpine grassland in Eastern Qilian Mountains, three species of grassland soil microbes (fungi, bacteria and antinomycete) were collected from the same study area (perennial cultivated grassland, inside the fence grassland, annual cultivated grassland and outside the fence grassland) in 2003, 2011 and 2017 usingplatelet count method. The results showed that:(1)At the same time, three types of soil microbes and nitrogen physiological populations were significant (p<0.05) in different disturbance habitats, and they decreased gradually with the increase of the soil depth; (2)In different time and different disturbance habitats, three types of soil microorganisms and the number of nitrogen physiological groups were significant (p<0.05; specifically, the ranks of soil bacteria, actinomycetes, nitrifying bacteria and aerobic nitrogen fixing bacteria distribution were 2003 > 2011 > 2017; and the ranks of fungi, denitrifying bacteria and anaerobic nitrogen fixing bacteria quantity distribution were 2017 > 2011 > 2003; (3) The total number of microorganisms in different disturbance habitat performance were 2003 > 2011 > 2017. Compared with 2003, the total number of microbes in 2011 and 2017 decreased by 1.65 and 2.86 times. The perennial grassland decreased by 3.12 and 5.12 times respectively, the grassland within the enclosure decreased by 3.05 and 4.09 times, and the grassland decreased by 2.11 and 3.26 times respectively. In conclusion, with the unreasonable grazing and artificial utilization, the number of soil microorganisms in the alpine grassland of East Qilian Mountains gradually decreased with time. Therefore, we should pay enough attention to grassland utilization and protection in this area.