The invention relates to a method for genetically transforming citrus mature internode stems, which comprises following steps: firstly, disinfecting mature materials, selecting semi lignified new shoots in a greenhouse, using sodium hypochlorite solution 5wt% to wipe the shoots once every two days for a week before the shoots are sampled, wiping for 3-4 times together, then, cutting the shoots which are wiped with the sodium hypochlorite solution, cutting the shoots into stems of 4-5cm under the aseptic condition, using the sodium hypochlorite solution 10wt% which is added with tween 2-3 drops to dip for 20-35min, washing with sterilized water for 3-4 times, cutting the shoots into the internode stems of 1-1.5cm to preserve for use, secondly, transforming, thirdly, regenerating resistant buds, fifthly, grafting for the second time in the greenhouse, sixthly, identifying the resistant buds and resistant plants, and adopting a public-known PCR analysis method to identify. The citrus mature materials of the invention have good disinfection effect and low contamination rate, the adventitious bud induction frequency reaches 48%, the grafting survival rate of the resistant buds reaches more than 80%, and regenerated transformed plants do not have chimerism. The method is in particular suitable for genetically transforming mature internode stems of main cultivars of citrus such as crystal sugar oranges, navel orange, Ponkan and the like.