The aim of this study was exploring the effects of plastic film mulching and application of water retaining agent combined with microbial fertilizer on quantity of soil microorganism, enzyme activity, and yield and water use efficiency of seed maize in Hexi Corridor. In oasis irrigation area of the Hexi Corridor, six treatments were set up, which were single plastic film mulching (AF), single application of super absorbent polymer (AW), single application of microbial fertilizer (AB), application of super absorbent polymer combined with microbial fertilizer (WB), plastic film mulching combined with microbial fertilizer (FB) and neither super absorbent polymer nor microbial fertilizer in open field (CK). The dynamic changes of soil microbial quantity, enzyme activity in 0-20 and 20-40 cm soil layer before sowing and after harvesting, and the yield of seed maize were analyzed. The results showed that:(1) Single or combined application of microbial fertilizer both could improve the activity of soil catalase, urease, invertase, phosphatase, and increase the number of fungi, bacteria and actinomycetes, and the content of soil microbial carbon and nitrogen, and improve the soil microbial environment. Among them, the effect of super absorbent polymer combined with microbial fertilizer was the best, and followed by the treatment of FB. (2) The treatment of AB had little influence on the growth of seed maize, but the treatment of FB could significantly increase the leaf area index and dry matter accumulation of seed maize, and could adjust the yield composition factors. (3) Different drought resistance measures and the combined application of microbial bacterial fertilizers could promote yield formation of seed maize. Among the different treatments, the grain yield of the FB treatment was the highest (10 105.64 kg/hm²), followed by the treatment of AF (8 967.24 kg/hm²) and WB (8 323.93 kg/hm²), and the production increased by 61.99%, 43.74% and 33.43% compared with CK, respectively. (4) The water use efficiency was the highest in the treatment of FB (2.40 kg/m³), followed by AF (2.15 kg/m³) and WB (1.89 kg/m³), and the water use efficiency significantly improved by 80.10%, 61.84% and 41.80% compared with CK, respectively. Therefore, comprehensive considering the yield, water production efficiency and soil microenvironment, the best combination of drought resistant measures combined with microbial fertilizer was plastic film mulching combined with microbial fertilizer, which could not only promote the growth and development of seed maize, but also improve irrigation water use efficiency and water use efficiency, meanwhile improve the soil micro-environment of the tillage layer, which was of great significance to the sustainable development of seed maize production in the Hexi Corridor.