Abstract: In order to understand the effects of exogenous diethyl aminoethyl hexanoate（DA-6） on the growth regulation and anthocyanin anabolism of alfalfa seedlings under UV-B radiation stress, the effects of leaf spraying or root application of 30 mg/L DA-6 solution on the growth, antioxidant capacity, photosynthesis, chlorophyll and anthocyanin anabolism of alfalfa seedlings under UV-B radiation stress(added UV-B 5 kJ/(m² ·d) on the basis of the natural light) were studied by nutrient solution sand culture method. After 15 days of treatment, the samples were taken for the determination of each index. The results showed that UV-B radiation stress significantly aggravated the cell membrane lipid peroxidation injury of cell membrane of alfalfa seedlings, and inhibited the growth, chlorophyll synthesis and photosynthesis of the seedlings. DA-6 solution by leaf spraying or root application could alleviate the stress of UV-B radiation on alfalfa seedlings in varying degrees, and DA-6 by root application had a better comprehensive alleviation effect, which increased the plant height, root length, shoot dry weight, root dry weight, and root-shoot ratio of the seedlings, improved the activities of superoxide dismutase, peroxidase and catalase in leaves, decreased the contents of superoxide anion, hydrogen peroxide and malondialdehyde, and promoted the net photosynthetic rate. DA-6 by root application promoted chlorophyll synthesis by ameliorating the degree of transformation obstruction from porphobilinogen to uroporphyrinogen Ⅲ in the chlorophyll synthesis pathway under UV-B radiation stress, and enhanced the key enzyme（phenylalanine ammonia lyase, chalcone synthase, chalcone isomerase, anthocyanin synthase） activities for anthocyanin anabolism such as phenylalanine ammonia lyase, thus promoting the synthesis and accumulation of anthocyanins. The results indicated that exogenous DA-6, especially root application, could alleviate the inhibitory effect of UV-B radiation stress on the growth and photosynthesis of alfalfa seedlings by enhancing antioxidant enzyme（peroxidase and catalase） activity and promoting chlorophyll and anthocyanin biosynthesis, and finally promoted the accumulation of dry matter in the shoot and root of the seedlings.