Abstract: Drought is one of the major abiotic stresses that severely limits plant growth and development, thereby restricting crop productivity. This study aims to isolate plant growth-promoting rhizobacteria (PGPB) with excellent growth-promoting and drought tolerance properties from the rhizosphere soil of Medicago sativa (alfalfa) in different arid environments, and to evaluate their effects on the growth, development, and physiological responses of Medicago sativa seedlings under drought stress. A total of 903 culturable bacterial strains were isolated using multiple media and identified through 16S rRNA gene sequencing. These strains predominantly belong to the phyla Proteobacteria, Actinobacteria, and Bacteroidetes, with Serratia and Pseudomonas being the dominant genera. A range of plant growth-promoting traits were detected in 82 rhizosphere bacterial strains, among which OvR165 (Enterobacter sp.), OvR548 (Acinetobacter sp.), and OvR687 (Pseudomonas sp.) exhibited notable phosphorus solubilization, nitrogen fixation, iron chelation, IAA secretion, and EPS production. Additionally, OvR548 and OvR687 demonstrated strong biofilm formation abilities. Pot experiments revealed that inoculation with these three strains significantly alleviated the inhibitory effects of drought stress on Medicago sativa seedlings. This led to improvements in plant height, root length, leaf area, and biomass accumulation, along with enhanced chlorophyll content, relative water content, and reduced membrane damage and reactive oxygen species accumulation. These results demonstrate the potential of these bacterial strains in mitigating drought stress and promoting plant growth. In conclusion, the rhizosphere of Medicago sativa in different arid regions of Qinghai harbors a rich and diverse reservoir of functional drought-tolerant PGPB, which holds significant ecological value and practical application prospects for sustainable grassland management and the development of microbial formulations in arid regions.