A rd29a cold inducible promoter was introduced into alfalfa (Medicago sativa L.) by Agrobacterium transformation. The GUS activity driven by the rd29a promoter increased in leaf and stem, but decreased in the root when the transgenic alfalfa was placed at 2\sp∘C. The GUS mRNA in stem tissue transiently increased within 2-3 days at 2\sp∘C. One or three copy number of a truncated rd29a promoter containing the dehydration responsive element was transcriptionally fused in two orientations with GUS and firefly luciferase genes respectively. These constructs were introduced into alfalfa leaflets by particle bombardment. In luciferase activity assay, the Dual Luciferase Assay System (firefly and Renilla luciferase) was used. The activities of firefly, Renilla luciferases, and GUS were unstable under cold treatment. The alfalfa response to the rd29a promoter was difference observed in Arabidopsis and tobacco. The Dual Luciferase Assay System was unsuitable for investigation at cold in alfalfa.