Abstract: Alfalfa root rot, a devastating soil-borne disease caused by complex pathogenic microorganisms, severely limits the stable and high-yield production of alfalfa and hinders the effective improvement of its forage quality. Identifying regionally dominant pathogenic strains and screening highly efficient biocontrol agents with targeted suppression capabilities are crucial strategies to achieve green, eco-friendly control of this destructive disease. This study focused on a typical high-incidence area of alfalfa root rot in Pingluo County, Ningxia Hui Autonomous Region, aiming to explore region-specific biocontrol solutions. By employing high-throughput sequencing technology to compare the rhizosphere microbial communities in the rhizosphere soils of healthy and diseased alfalfa plants, this research systematically revealed the distinct characteristics of rhizosphere microbial imbalance during the progression of alfalfa root rot. Based on these microbial community analysis results, the pathogenic fungus Paraphoma rhaphiolepidis, which is highly associated with the occurrence of alfalfa root rot in this area, 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 screened from the rhizosphere soil of healthy alfalfa plants, and was ultimately identified as Bacillus velezensis through molecular and physiological-biochemical identification. In vitro antagonism tests clearly demonstrated that Bacillus velezensis ARF-SR2 exhibited remarkably significant antagonistic activity against the target pathogen P. rhaphiolepidis, achieving 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 could significantly disrupt 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 ARF-SR2 possesses multiple beneficial functional traits, including nitrogen fixation, phosphorus solubilization, secretion of various hydrolytic enzymes, siderophore production, and indoleacetic acid (IAA) synthesis, coupled with a strong ability to form biofilms. These traits endow the strain with excellent rhizosphere colonization capacity and substantial plant growth-promoting potential. Pot experiments further validated the prominent biocontrol efficacy of ARF-SR2, achieving 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 to 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 exhibits extremely promising application prospects in the green control of alfalfa root rot in Ningxia through a synergistic combination of multiple mechanisms, including targeted disease suppression, efficient plant growth promotion, and stable rhizosphere colonization. This strain can serve as a core functional microorganism for the engineered development of integrated microbial agents integrating “disease control and growth promotion” functions.