The Salt Overly Sensitive (SOS) signal transduction pathway is pivotal in Na⁺ efflux and facilitates ion transport and homeostasis for improved salt tolerance in plants. Ten alfalfa varieties were used as experimental materials and two alfalfa regeneration systems were established and optimized. Cotyledons and hypocotyls were initially used as explants to induce embryogenic callus via the indirect production of somatic embryos to establish a callus acceptor system. Cotyledonary nodes were used as explants to induce adventitious bud formation via direct organogenesis, thereby establishing an in vitro regeneration system that could be used for the genetic transformation. Agrobacterium-mediated transformation of the cotyledonary nodes of the alfalfa ‘Golden Empress b’ was used to generate 25 independent sources of transformed plants exhibiting herbicide tolerance. Four of the positive transgenic plants were randomly selected for southern blot analysis, and three hybridization signals with one or two copies were detected. Reverse transcription polymerase chain reaction showed that the Bialaphos resistance (Bar) and SOS1 genes were expressed in transgenic plants and that multiple exogenous salt-tolerant genes were integrated into the transgenic plant genome and expressed at the transcriptional level. The overexpression of Arabidopsis SOS genes in alfalfa conferred a high degree of salinity tolerance, enhanced plant growth, lowered the accumulation of Na⁺, increased the accumulation of K⁺ in the leaves, and altered physiological and biochemical parameters in response to salt stress.