In order to reveal the effects of different nitrogen application rates on soil microorganisms in cotton fields，a field experiment was conducted with in Alaer region for three consecutive years from 2018 to 2020.Six nitrogen treatments，including 0 kg/hm²(N0)，90 kg/hm²(N1)，180 kg/hm²(N2)，270 kg/hm²(N3)，360 kg/hm²(N4)and 450 kg/hm²(N5)，were set up to study the effects of located and quantitative nitrogen fertilization on the number and structure proportion of the three microflora in 0～60 cm soil layer.The results showed that，in the 0～60 cm soil layer of each treatment，soil microorganisms were mainly bacteria，actinomycetes followed，and fungi were the least.Continuous located and quantitative nitrogen fertilization increased the total numbers of soil bacteria，fungi and actinomycetes in 0～60 cm soil layer.With the increase of nitrogen application rates，the total numbers of soil bacteria，fungi and actinomycetes basically increased first and then decreased.The maximum number of bacteria and actinomycetes appeared at the treatment N3，and the changes were mainly reflected in 0～40 cm soil layer(bacteria from 0～40 cm soil layer accounted for 71.92%～79.65% of the total，actinomycetes from 0～40 cm soil layer accounted for 77.21%～92.06% of the total)；The maximum number of fungi appeared at the treatment N4，and the variation was mainly reflected in 0～20 cm soil layer(fungi from 0～20 cm soil layer accounted for 35.62%～65.84% of the total).With the increase of nitrogen application rate，the total number of the three microflora increased first and then decreased，the maximum values appeared at the treatment N3(9.70×106 CFU/g)，and the bacteria/fungi and actinomycetes/fungi basically showed a decreasing trend，while the proportion of fungi generally showed an increasing trend.Therefore，the recommended nitrogen application rate in Alar region is 270 kg/hm²，which can balance the structure of soil microbial flora and obtain a better soil ecological environment.