The study focused on the dynamics of MDA contents and the activities of protective enzymes in the leaves of alfalfa varieties with various resistance to Aphis medicaginis Koch. The results indicated that the MDA contents of susceptible varieties were higher than that of the resistant ones when their leaves were piercing-sucked by the aphid. Throughout the period of the aphid attacks, the MDA contents displayed a rising trend in both of the susceptible and resistant varieties. The SOD, POD and PAL activities in the susceptible varieties appeared lower than that of the resistant ones. The activities of SOD and POD of all varieties rose firstly and then declined. However, the PAL activity rose to a peak and remained high during the testing period. The CAT activity formed cycles of rising and declining, with the activity peaks and bases showing opposite positions in the resistant and susceptible varieties, respectively. The PPO activity of susceptible varieties became higher compared to resistant ones in the earlier period but lower in the later part. Therefore, under the stress of the aphid, the contents of MDA and the activity of SOD, POD, PAL and PPO in susceptible and resistant varieties are closely related to the aphid-resistance of alfalfa. These biochemical markers may be used as physiological index for aphid-resistance appraisal. However, the relationship between CAT and aphid-resistance of alfalfa is worth of further research.