In order to explore the influences of combined application of sheep manure-derived organic fertilizer and chemical fertilizer on soil microbial community structure and soil fertility in tobacco-growing area of Luoyang, a pot experiment was conducted and high-throughput sequencing techniques was used to study the differences in community structure and diversity of soil bacteria and fungi under four fertilization treatments, no fertilizer (CK), 100% inorganic nitrogen fertilizer (T0), 50% sheep manure organic nitrogen fertilizer +50% inorganic nitrogen fertilizer (T50), and 100% sheep manure organic nitrogen fertilizer (T100). And the relationship between soil physiochemical properties and soil microbial diversity were analyzed. And the results showed that the dominant soil bacteria phyla were Proteobacteria, Acidobacteria and Actinobacteria, while the dominant soil fungi phyla were Ascomycota and Basidiomycota. Compared with other treatments, T50 had the highest relative abundances in Proteobacteria, Acidobacteria and Ascomycota and the lowest relative abundances of in Actinobacteria and Basidiomycota. NMDS analysis and similarity analysis showed that soil microbial community structure of T50 was significantly different from those of other treatments. Alpha diversity analysis also showed that T50 had the highest richness and diversity of bacterial and fungal communities. Soil physicochemical properties and soil key enzyme activities were significantly different under the four treatments, and T50 had the highest soil nutrient contents and soil carbon and nitrogen metabolizing enzyme activities. Pearson correlation analysis showed that soil alkali-hydrolyzed nitrogen, available phosphorus, available potassium, protease and urease activities were most closely related to soil microbial diversity. In summary, combined application of sheep manure-derived organic fertilizer and chemical fertilizers can promote the release and transformation of soil nutrients by affecting the community structure and diversity of soil bacteria and fungi, which are conducive to the supply of soil nutrients and the improvement of soil quality. The results provide a theoretical basis for the application of sheep manure-derived organic fertilizer to improve soil quality in tobacco-growing areas of Luoyang.