The physiological characteristics and salt tolerance mechanism of two genotypes of alfalfa (Medicago sativa L.) Adrenalin (salt-stress tolerant) and Peru (salt-stress sensitive) under salt stress were explored by using 150 mmol/L NaCl to deal with two genotypes of alfalfa seedlings. Catalase (CAT), malondialdehyde (MDA), peroxidase (POD), relative water content (RWC), Chlorophyll (Chl), and proline (PRO) were respectively determined before and after 2, 4, 6, 8, 16 hour of salt stress. The result showed that the catalase activity, the peroxidase activity, and the malondialdehyde content of two genotypes of alfalfa showed a trend of increase under salt stress, while the proline content and the relative water content showed an opposite trend. The chlorophyll content significantly decreased. In addition, it was found that the variation of the relative water content and the chlorophyll content corresponded to the change of plant phenotype. Principal component analysis showed that the catalase and the malondialdehyde contributed the most, which could provide a theoretical basis for the research on salt tolerance mechanism and molecular breeding of alfalfa.