Aiming at the problems of serious soil desertification and continuous reduction of effective soil nutrients in the agricultural and animal husbandry interlaced arid area in Inner Mongolia, this study used Aohan alfalfa, old awn wheat, and fodder oats at the Siziwang Banner, Ulanchabu City, Inner Mongolia Autonomous Region. There were a total of five treatments, namely, single seeding alfalfa, single seeding old awn wheat, single seeding fodder oats, alfalfa and old awn wheat mixed sowing, and control. The results showed that the urease activity and microbial biomass nitrogen content of mixed planting were higher than other treatments. With the advancement of the growth period, the bacteria α-diversity index showed an upward trend; β-diversity analysis showed that planting method was the main factor affecting bacteria diversity and sampling time was the main factor affecting fungi diversity. In summary, mixed planting treatment was more helpful to improve soil enzyme activity, microbial biomass, and enrich soil microbial diversity, which was of great significance to maintain the balance of soil ecosystem. It is hoped that this study will contribute to the theoretical basis and practical experiences for efficient utilization of microbial resources in the field of soil improvement.