The invention discloses a method for improving conjugational transfer efficiency of fluorescent plasmid transfer of fluorescence labeling strains of rhizobia by taking sterilizing alfalfa leaves as triparental mating carriers instead of sterile filter membranes. The method can be used for efficiently constructing CFP (cyan fluorescent protein) gene labeled strains of rhizobia. The method specifically comprises the following steps of taking small and tender leaves of alfalfa, uniformly putting the leaves in a single layer in a culture dish after washing the leaves with distilled water, and sterilizing the leaves; taking out the leaves with a pair of sterile tweezers, sticking the leaves to the surface of TY culture media, and covering the culture dish; and using the leaves to replace microporous filter membranes in the triparental mating process to carry out mixed growing culture on donors, assistant bacteria and recipient bacteria. Compared with the microporous filter membranes as the mating carriers, the alfalfa leaves, as the mating carriers, can improve the conjugational transfer efficiency of the fluorescent plasmids by 8.4-11 times, and the difference is obvious. As the used raw materials are alfalfa plant leaves which are renewable and accessible, the method has the advantage that the raw materials are cheap and accessible, is simple and convenient to operate and is suitable for constructing the fluorescence labeling strains of rhizobia.