Establishment of safe and effective gene vectors and in vivo delivery systems are crucial for gene therapy. The vectors for gene therapy comprise viral and non-viral vectors. Of which the viral vectors have been widely applied in clinical trials, due to their highly transduction efficiency; while they still encounter many potential safety concerns. In contrast, the non-viral vectors are safe and easy to prepare gene vehicles. However, the insufficient transfection efficiency limits their clinical application. Many efforts have been devoted into optimizing the delivery systems to enhance the gene transfer efficiency of non-viral vectors, a few of which are currently undergoing clinical trials. In this review, the recent advances of non-viral gene delivery systems and their clinical prospects were summarized.