Alfalfa root rot is one of the devastating soil-borne diseases caused by complex pathogenic microorganisms. It has severely limited the stable and high-yielding, as well as the forage quality of alfalfa. It is often required to identify the regionally dominant pathogenic strains and then screen highly efficient biocontrol agents with targeted suppression, particularly for the eco-friendly control of the destructive disease. This study aimed to explore the region-specific biocontrol solution in the typical high-incidence area of alfalfa root rot in Pingluo County, Ningxia Hui Autonomous Region, China. High-throughput sequencing was employed to compare the rhizosphere microbial communities in the rhizosphere soils of healthy and diseased alfalfa plants. A systematic investigation was also conducted to characterize the rhizosphere microbial imbalance during the progression of alfalfa root rot. According to the microbial community analysis, the pathogenic fungus, Paraphoma rhaphiolepidis, which was highly associated with the occurrence of alfalfa root rot, was successfully isolated and accurately identified for the first time in this specific region. Furthermore, a highly efficient antagonistic bacterium strain, designated as ARF-SR2, was isolated and then screened from the rhizosphere soil of healthy alfalfa plants, ultimately identified as Bacillus velezensis after molecular and physiological-biochemical identification. In vitro antagonism tests demonstrated that Bacillus velezensis ARF-SR2 exhibited remarkably significant antagonistic activity against the target pathogen P. rhaphiolepidis, indicating an inhibition rate of 68.81%, while the minimum inhibitory concentration (MIC) of its fermentation supernatant was determined to be 1 mg/mL. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) observations further confirmed that the fermentation supernatant of ARF-SR2 significantly disrupted the mycelial morphology and cellular ultrastructure of P. rhaphiolepidis, eventually leading to the dissolution of the pathogen’s cell wall, condensation of cytoplasmic contents, and disintegration of internal organelles. Functional characterization assays indicated that the ARF-SR2 also shared multiple beneficial functional traits, including nitrogen fixation, phosphorus solubilization, secretion of various hydrolytic enzymes, siderophore production, and indoleacetic acid (IAA) synthesis. A strong performance was coupled to form biofilms. There was a strain with the excellent rhizosphere colonization and substantial potential for plant growth. Pot experiments further validated that the biocontrol efficacy of ARF-SR2 achieved a control efficiency of 64.90% against alfalfa root rot. Notably, the strain significantly alleviated the growth inhibition of alfalfa induced by P. rhaphiolepidis stress, as key growth indicators, such as plant height, root length, fresh weight, and dry weight, increased by 36.87%-93.44% (P<0.05), compared with the pathogen-infected control group. Simultaneously, ARF-SR2 treatment significantly elevated the soluble protein content and antioxidant enzyme activities (including SOD and CAT) in alfalfa plants, thereby enhancing the plant's stress tolerance and disease resistance. In summary, the strain Bacillus velezensis ARF-SR2 exhibited extremely promising prospects in the green control of alfalfa root rot in Ningxia. A synergistic combination of multiple mechanisms included the targeted disease suppression, efficient plant growth promotion, and stable rhizosphere colonization. This strain can serve as the functional microorganism for the engineered microbial agents, thereby integrating “disease control and growth promotion”.