To establish an efficient Agrobacterium rhizogenes-mediated transformation system of Pueraria lobata, in this study, tissue cultured seedlings were used as explants to explore the effects of different factors on efficiency of Agrobacterium rhizogenes-mediated transformation system of P. lobata. The results indicated that genotype was the most important limiting factor of all. A. rhizogenes K599 was identified to be the most suitable strain. The optimal explant material was immature leaves that had just unfolded from the first to second nodes of the 5th to 13th generation tissue culture seedlings subcultured for 8 days. After 3 days of pre-culture and 15 minutes of bacterial infection, the highest induction rate of hairy roots could up to be 22.4%. The optimal culture method for proliferation of hairy roots in P. lobata was solid medium culture, and the fresh weight of hairy roots grown on solid medium was 75 times that of hairy roots grown on liquid medium. The PCR analysis and fluorescence microscopy assays showed the expression of GFP and rolB gene in the hairy roots of P. lobata was stable, and the rate of co-transformation was 80%. In our study, an A. rhizogenes-mediated genetic transformation system of P. lobata was preliminarily established, which laid a foundation for gene function identification in P. lobata.