In order to reveal the after-effects and superimposed effects of annual nitrogen application rate on soil microbial flora of reseeding soybeans, a split-plot experiment was conducted in 2017—2019 under wheat soybean rotation, in which the annual nitrogen application included 0 (N0), 104 (N1), 173 (N2) and 242 (N3) kg/hm² in wheat season, while 0 (S0), 69 (S1), 138 (S2) kg/hm² in reseeding soybean season, then the changes of the number of soil bacteria, fungi and actinomycetes, the structure of soil flora and the number of nitrogen physiological groups in soybean stubble were analyzed. The results show that nitrogen application in the wheat season has a significant after-effect on the number of soil microorganisms in reseeding soybean. Under the condition of no nitrogen application in reseeding soybean, the range of N2 to N3 nitrogen application in wheat season is beneficial to increase soil bacteria, fungi, actinomycetes and nitrogen physiological groups. Under the condition of no nitrogen application in wheat season, nitrogen application in soybean stubble significantly increases the number of three types of soil microorganisms and nitrogen physiological groups, but the higher nitrogen application in soybean stubble significantly reduces ammoniating bacteria and aerobic autogenous nitrogen-fixing bacteria. Nitrogen treatment in wheat season combined with S1 in soybean stubble increases the three types of soil microorganisms, B/F and A/F values, ammonifying bacteria, nitrogen-fixing bacteria and nitrifying bacteria compared with combined with S2, among of which, N1S1 is the highest in soil actinomycetes, aerobic autogenous nitrogen-fixing bacteria, B/F and A/F values, while N2S1 is near the highest in soil bacteria, fungi and ammonifying bacteria. Excessive annual nitrogen application increases soil denitrifying bacteria, significantly reduces B/F and A/F values, and inhibits three soil microorganisms and other nitrogen physiological groups to varying degrees. Therefore, proper amount of nitrogen application for wheat and subsequent soybeans is an important measure to maintain the dominant soil flora, the optimal application of annual nitrogen fertilizer should fully consider the superimposed effects of nitrogen fertilizer of the preceding and subsequent crops. Under the conditions of this study, the combination of 104-173 kg/hm² (N1 to N2) nitrogen in wheat season and 69 kg/hm² (S1) nitrogen fertilizer in soybean season is beneficial to soil microbial survival, soil fertility and quality, and ecological environment of farmland.