High-frequent regeneration is essential for plant genetic transformation.To further improve regeneration efficacy of white clover（Trifolium repens L.）,different concentration and combination of phytohormones of 2,4-D,KT,6-BA and NAA were investigated on shoots induction and proliferation from the cotyledon.And acclimation of the regenerated plantlets was also optimized.The results showed that better-quality of the shoots cluster can be induced from the cotyledon when the explants grew on the MS induction medium supplemented with 0.5 mg·L^-1 6-BA and 0.1 mg·L^-1 NAA.Besides,about 90% of shoots could take roots with an average of more 6 adventitious roots for every green shoot after four-weeks culture on 1/2MS medium containing 0.2 mg·L^-1 IBA.Moreover,over 85% of the regenerated plantlets survived when transplanted on the mixture of the turf and pearlite（2:1）.Our progress will do good help to genetic transformation system of Leguminosae plants,genetic improvement and germplasm innovation of Trifolium repens L.