In recent years, due to the continuous application of chemical fertilizers, soil fertility in tobacco fields has been declining, and seriously restricting the improvement of tobacco yield and quality. Distillers' grain (DG) is the main by-product of Chinese spirits production, which can be composted to produce organic fertilizer with the advantages of improving soil fertility and crop yield and quality. However, the application of DG organic fertilizer to flue-cured tobacco and its effects on soil microbial community has been rarely reported to date. In this study, we investigated the effects of DG organic fertilizer on soil chemical properties, fungal community composition and structure, and the relationships between them. The results showed that applying DG organic fertilizer altered the composition and diversity of fungal community at different growth periods of tobacco. Ascomycota, Mortierella and Basidiomycetes are the dominant phyla in soils. Each of the soil chemical properties is able to explain to the fungal community variation between CK and DG organic fertilizer treatments to some extent, and soil available phosphorus was the key factor driving the changes of soil fungal community. The results of this study will play a vital role in facilitating comprehensive promotion in tobacco-growing field and improving the yield and quality of tobacco.