Medicago sativa L. is a perennial legume forage. In order to analyze the function of UFO gene from Medicago sativa, we isolated the full-length coding sequence by PCR amplification in alfalfa variety ‘Zhongmu No. 1’. The plant expression vector 3302-3flag-UFO was constructed by DNA recombination technology, and transgenic alfalfa was obtained by deployment of Agrobacterium tumefaciens mediated transformation method. The transgenic efficiency of alfalfa was improved by changing the ratio of hormone concentrations and improving plant expression vector. The type and ratio of hormones have resulted in a difference on the formation of resistant calli and the degree of vitrification of calli. By taking use of optimal transformation strategy, a total of 67 resistant callus were generated, while 56 young alfalfa regenerated plants were obtained. Finally, 17 mature regenerated plants were obtained for seed propagation. Taking together, this study generated the transgenic plants with UFO overexpressing vector, which might provide materials for further functional study of UFO gene.