Organic fertilizer application is an effective approach to enhancing soil organic matter, soil biological activity as well as crop yield in agriculture. In the present research, a two-year field experiment was established to study the impacts of combined use of organic fertilizer with chemical fertilizer (CF) on cotton yield, soil microbial biomass, soil enzyme activities and soil microbial community structural composition in drip-irrigation condition in northern Xinjiang. Compared with CK, 100% chemical fertilizer treatment (300 kg N/hm², 90 kg P₂O₅/hm², 60 kg K₂O/hm²) or treatments of organic manure at different rates combined with chemical fertilizer significantly increased (P < 0.05) cotton yield. With chemical fertilizer or organic manure supply, cotton yield could be reached up to 4945-4978 kg/hm², however, there was no statistical difference (P < 0.05) among 100% CF fertilization and treatments with 20% to 40% chemical fertilizer substituted by 3000-6000 kg/hm²organic fertilizer. The number of soil bacteria, actinomycetes as well as pseudomonas significantly increased with increasing amendment rate of organic manure, but the number of soil fungi decreased with organic fertilizer rates increasing. For instance, the number of soil bacteria increased by 13%-41% in the treatments of organic manure at different rates combined with CF than in the treatment with 100% CF, whereas, the number of soil fungi in the treatment with 100% CF fertilizer is 1.4-1.6 times higher than that in the treatment with organic manure combined with CF at budding stage of cotton. Organic fertilizer application significantly enhanced (P < 0.05) soil enzyme activities. Compared with the treatment with 100% CF supply, soil enzyme activities of alkaline phosphatase, fluorescein acetate esterase, β-glucosidase and dehydrogenase increased by 3.8%, 17%, 18% and 55% in the treatments with 80% CF combined with 3000 kg/hm² organic manure, and increased by 10.1%, 19%, 20%, and 60% in the treatments with 60% CF fertilizer combined with 6000 kg/hm² organic manure (BF-organic), respectively. The ratios of soil bacteria/fungi, soil actinomycetes/fungi and pseudomonas/fungi increased with increasing application rate of organic manure, but pseudomonas/bacteria ratio markedly reduced in the treatment with 100 CF or organic fertilizer supply. A significant positive correlation (P < 0.05) was observed between the number of soil bacteria, actinomycetes and pseudomonas was significantly positively correlated with the activity of soil enzymes including dehydrogenase, β-glucosidase, alkaline phosphatase and fluorescein acetate esterase, respectively. However, an obvious negative correlation was observed between soil fungi number and the activity of soil enzymes such as dehydrogenase, β-glucosidase, alkaline phosphatase and fluorescein acetate esterase. Our conclusion is that 20%-40% of total chemical fertilizer application rate can be substituted by 3000-6000 kg/hm² organic fertilizer with no cotton yield decrease. Moreover, organic fertilizer addition addition plays a significant role in increasing soil enzyme activities, regulating soil microbial community structural composition as well as improving soil biological properties in cotton mono-cropping system under drip-irrigation condition in northern Xinjiang.