Abstract: Following the development of plants resistance and transgenic techonology, breed of new resistant alfalfa by transforming heterogenous gene has being become more and more remarkable to pepole.So,establishing a high-frequence system of regeneration plants is the base of high efficiency transform for heterogenous gene. Cotyledon, hypocotyl and root of 5-7 day-old seedling of Xinjiang big leaf alfalfa were selected as explants. MS and 8 groups of 2,4-D with different concentrations from 0.1mg/L to 3mg/L were used to induce callus, then MS and 2,4-D2mg/L and 10 groups of KT with different concertrations from 0.01mg/L to 0.5mg/L were used to induce callus; the shoots were induced in the medium of MS with additional 6-BA 0.5mg/L and NAA 0.05mg/L; MS was used to produce roots. Results showed that: callus tissues had much better growing status and could be used to produce differentiation shoots in medium of MS with 2,4-D 2.0mg/L and KT 0.2mg/L, the averaging calli frequences were 93.1%, 100% and 100% for cotyledon, hypocotyl and root; After growing 40-80 days in the medium of inducing regeneration shoot, callus tissues which originated from cotyledon, hypocotyl and root produce regeneration shoots and its averaging regeneration shoots frequence was 50%, 78% and 50% respectively; when shoots grew to about 2cm height, it was transferred into medium of MS to root; afer 14 days, regeneration plants were transferred into flower-soil and its survivable rate was more than 90%. All of cotyledon, hypocotyl and root can obtain regeneration plants in this culturing system. Moreover root can act as a good material of explant to obtain regeneration plant while researches in this field are still poor.