Abstract: This study aims at comparing the effects of different mutagenesis treatments on alfalfa. 60Co-γ rays, ultraviolet and EMS were used for induced mutation of four alfalfa cultivars’dry seeds. The contemporary materials were taken as research objects, the influence of mutagenic treatment on the physiological and biochemical indexes of alfalfa was explored. After mutation treatments, the activity of three anti-oxidative enzymes showed an increasing trend. The effect of 60Co-γ irradiation on SOD was accelerating, ultraviolet treatment reduced SOD activity, EMS treatment on SOD showed low- concentration promotion and highconcentration inhibitory. Along with the increasing of 60Co-γ irradiation, POD and CAT activity decreased, while POD and CAT activity increased with the increasing ultraviolet dosage. The effect of EMS treatment was different in different varieties. The effects of mutagenic treatments on soluble protein, soluble sugar and free proline in alfalfa were different due to dosage and variety, and did not affect MDA content obviously. The mutation treatments improved the activities of three anti-oxidative enzymes, 150 Gy 60Co-γ, 90 min ultraviolet and 0.4% EMS treatment had significant effect on the physiological activity of alfalfa.