Drought stress is one of the leading impediments that limit the productivity of global alfalfa (Medicago sativa). The underlying molecular and genetic mechanisms for drought tolerance in alfalfa remain largely unclear. In order to fully reveal the transcriptional changes of alfalfa in response to abiotic stress, the alfalfa transcriptome database under mannitol (simulated drought stress), NaCl (simulated salt stress), or exogenous ABA application was built via various RNA-seq technologies. Through further screening of the transcriptome database, a number of genes significantly induced by drought stress, such as the Nuclear Transport Factor 2-like (MsNTF2L), Drought-Induced Unknown Protein 1 (MsDIUP1), and MsNST1, were identified. These three genes were transferred into alfalfa by overexpression and RNAi techniques, and their physiological characteristics and transcriptional level response were synthetically studied. Alfalfa MsNTF2L-OE plants have been approved by the Ministry of Agriculture of China to carry out the field test in Gansu Province. Furthermore, we constructed a GWAS population and obtained 50 excellent plants with strong drought tolerance and high hay-yield. These studies provide a theoretical foundation for drought-tolerant molecular breeding of alfalfa