Biological nitrogen fixation provides a natural source of nitrogen for agro-ecosystems. Exploring the effects of long-term organic fertilization regime on soil diazotrophic communities can provide scientific basis for rational application of organic fertilizer and maintenance of soil diazotrophic community diversity. In this study, the long-term fertilization experiment under wheat-sweet potato system has been established in an alkaline soil since 1981. Soil samples were collected from without fertilizers control (CK), chemical fertilizer alone (NPK), and chemical fertilizer plus organic fertilizer (NPKM). The Illumina Miseq high throughput sequencing technologies was employed to study soil diazotrophic community composition, diversity, and its relationship with soil environmental factors. The results showed that long-term (37 year) application of organic fertilizer decreased the diversity and richness of soil diazotrophic community compared to CK and NPK treatment. The richness index Chao1 and observed species was significantly positively correlated with pH, and significantly negatively correlated with soil organic carbon, total nitrogen and soil nutrients (i.e., NO₃^--N, available phosphorus and available potassium). Principal coordinate analysis (PCoA) indicated that long-term fertilization significantly altered community structure of soil diazotrophic, and the application of organic fertilizers had a greater impact on the structure of diazotrophic than application of chemical fertilizers alone compared to without fertilizer control. Redundancy analysis (RDA) suggested that soil organic carbon and available potassium were main influential factors on changes of diazotrophic community structure. Long-term addition of organic fertilizer reduced the relative abundance of Proteobacteria, Cyanobacteria, Betaproteobacteria and Azoarcus, and increased the relative abundance of Nitrospirae, Acidobacteria and Nitrospira, which was closely related to soil pH, organic carbon and available nutrients. Therefore, this study revealed that organic fertilizer addition had a greater change than chemical fertilizer alone on diazotrophic community structure, and it increased also the inhibition of diazotrophic community diversity.