This study aimed to improve the Agrobacterium-mediated transformation system of Rehmannia glutinosa L. by optimizing the factors that influence the transformation efficiency. Through orthogonal experiment, conditions affecting Agrobacterium mediated transient transformation of leaf discs were evaluated, and the transient GUS assay was performed to observe the transformation efficiency. The results showed that the co-cultivation time was the most significant factor influencing the transformation efficiency, followed by pre-cultivation time, bacteria concentration and infection incubation time. The optimized Agrobacterium mediated transformation procedure for R. glutinosa was as follow: leaf discs without pre-culturing were infiltrated with A. tumefaciens for 5 min at optical density (OD600) 0.6 in liquid MS medium, then the infected explants were co-cultivated with A. tumefaciens in shoot induction medium containing l00 μM acetosyringone in dark for 3 days. Under optimal conditions, the highest transient expression efficiency of GUS was 65.63%. An effective Agrobacterium mediated transformation system was established for R. glutinosa by optimizing the influencing factors.