This study focused on alfalfa (Medicago sativa cv. Xinmu No. 4) as the experimental material, and a two-year field plot controlled experiment was conducted to compare the effects of different co-application ratios of organic and chemical fertilizers on alfalfa yield, soil nutrient status, and soil biological characteristics. The six fertilization treatments were as follows: CM0 (100% cattle manure), CM1 (75% cattle manure + 25% chemical fertilizer), CM2 (50% cattle manure + 50% chemical fertilizer), CM3 (25% cattle manure + 75% chemical fertilizer), CM4 (100% chemical fertilizer), and CK (no fertilizer application). The results showed that alfalfa hay yield was highest under the CM3 treatment in both 2024 and 2025, representing increases of 38.03% and 40.85%, respectively, compared with the control (CK). Relative to the other treatments, CM3 significantly increased soil total nitrogen, alkali-hydrolyzable nitrogen, available phosphorus, readily available potassium, and organic matter contents. In addition, CM3 markedly enhanced the activities of soil nitrate reductase (NR), nitrite reductase (NiR), and the root enzymes glutamate synthase (GOGAT) and glutamine synthase (GS). The combined application of organic and chemical fertilizers significantly reshaped the soil bacterial community structure associated with alfalfa. Under the CM3 treatment, Chao1, Shannon, and ACE indices of soil bacterial diversity increased, whereas the Simpson index decreased. Moreover, the CM3 treatment was associated with higher relative abundances of several key bacterial phyla and genera. The 25% cattle manure plus 75% chemical fertilizer (CM3) treatment exhibited the strongest overall effects, significantly increasing total alfalfa hay yield, enhancing soil macronutrient availability and key enzyme activities, improving soil microbial α-diversity, and optimizing soil bacterial community structure. This treatment consistently outperformed the no-fertilizer control (CK) and all other organic–inorganic fertilizer combinations. Collectively, these findings provide robust scientific evidence supporting strategies to increase forage productivity, mitigate environmental impacts, and promote the sustainable development of the grassland industry.

Global demand for alfalfa, a vital forage resource for ruminants, is growing. Currently, ruminant production is expanding rapidly in parts of Asia and Africa, further driving demand for high-quality alfalfa forage. Nevertheless, long-term indiscriminate fertilization and imbalanced nutrient input ratios in alfalfa production not only lower fertilizer use efficiency and crop productivity but also induce a range of soil degradation and environmental issues, thereby constraining the sustainable development of the forage-livestock industry. In this study, the alfalfa cultivar ‘Xinmu No. 4’ (Medicago sativa cv. ‘Xinmu No. 4’) was used as the experimental material. A two-year field block experiment was performed to investigate the effects of different organic–inorganic fertilizer ratios on the nutrient use efficiency of alfalfa, with six fertilization treatments: CM0 (100% cow manure), CM1 (75% cow manure + 25% chemical fertilizer), CM2 (50% cow manure + 50% chemical fertilizer), CM3 (25% cow manure + 75% chemical fertilizer), CM4 (100% chemical fertilizer), and CK (no fertilization application). The results demonstrated that the CM3 treatment (25% cow manure combined with 75% chemical fertilizer) significantly enhanced nitrogen, phosphorus, and potassium accumulation in alfalfa relative to the CK, sole cow manure (CM0), and sole chemical fertilizer (CM4) treatments (p < 0.05). Additionally, plant nutrient accumulation in 2025 was markedly higher than that in 2024 across treatments (p < 0.05). Combined organic and inorganic fertilization substantially improved the agronomic use efficiency of nitrogen, phosphorus, and potassium in alfalfa, with the CM3 ratio exhibiting the most pronounced beneficial effect. For alfalfa cultivation in Xinjiang, the combined application of 25% cow manure and 75% chemical fertilizer is recommended to optimize nutrient accumulation and utilization efficiency, ultimately boosting alfalfa yield and quality.