Nonviral vectors have been attracting more attention for several advantages in gene delivery and the development of nonviral gene carriers with high delivery efficiency and low cytotoxicity has long been a key project. Starburst polyamidoamine dendrimers are a class of synthetic polymers with unique structural and physical characteristics. However, when they are used as gene carrier, the gene transfection efficiency is not satisfactory. Herein, a novel thioketal-core polyamidoamine dendrimer (i.e., ROS-PAMAM) was synthesized and characterized. Compared to ethylenediamine-core dendrimers or widely used cationic polymers of polyetherimide, ROS-PAMAM showed lower cytotoxicity. Moreover, ROS-PAMAM demonstrated reactive oxygen species responsive characteristics, which can facilitate the release of siRNA in the tumor microenvironment. In vitro gene transfection experiments based on A549 cells confirmed that siRNA/ROS-PAMAM exhibits high gene transfection efficiency. It is concluded that ROS-PAMAM shows great potential as a generalizable vehicle for gene therapy applications.