In the present study，0～150 kg·hm-2 five different application levels of nitrogen fertilizer were set up to study their effects on the yield of alfalfa and the correlation between fungal diversity and physicochemical properties in the rhizosphere soils by using the ITS high-throughput sequencing and the correlation analysis methods.The results showed that the yields of N60 and N90 were significantly higher than those of the other treatments，meanwhile，the important soil quality indicators，i.e.， total nitrogen (TN) and soil organic matter (SOM) were both higher.The fungal species richness (Chao1 and PD) and whole fungal diversity (Shannon) were all significantly higher in N60 and N90 compared with N0，N120，and N150.Further，by weighted unifrac PCoA analysis and three complementary non-parametric multivariate statistical tests (Adonis，Anosim，and MRPP)，samples from the same treatment were clustered together and the difference between paired groups was all significant (P<0.05).Ascomycota was the major phylum (92.77%±2.50%) across all the samples，while on the Order level，N120 and N150 showed a much more similar fungal composition.By using co-occurrence network analysis，it showed that N60 and N90 both had higher community complexity and stability (exhibited by several nodes and cohesion，and the ratio of Mutual Exclusion:Copresence).Moreover，combined with LEfSe and RDA analysis，it was found that the biomarkers at the genus level of each sample mainly contributed to the overall responses to the physiochemical properties of rhizosphere soils.Collectively，the yield，physicochemical properties，and fungal diversity of alfalfa were all significantly better in N90.But most of the biomarkers were belonged to the pathogenic bacteria，suggesting that long-term treatment of alfalfa tillage soil under N90 nitrogen application concentration may promote the enrichment of soil pathogenic bacteria. In conclusion，the yield of alfalfa and the diversity and stability of the fungal community were significantly improved under appropriate nitrogen treatment，and the fungal community structure changed significantly when the nitrogen treatment exceeded a certain threshold.At the same time，for the selection of nitrogen concentration，not only the overall stability of alfalfa yield and soil fungal community should be considered，but also the accumulation of soil potential pathogenic bacteria under a specific concentration for a long time should be considered.