RNA interference (RNAi) has potential advantages over other gene therapy approaches due to its high specificity and the ability to inhibit target gene expression. Thus, developing a stable, safe, effective siRNA delivery system is necessary to achieve targeted therapy. Here, we developed such a system by conjugating gelatin-based nanoparticles with the nucleolin-targeted AS1411 aptamer and siRNA sequence together (Apt-GS/siRNA NPs) via a disulphide linker. These Apt-GS/siRNA NPs demonstrated favourable release of siRNA under reducing conditions owing to disulphide cleavage. Furthermore, the Apt-GS/siRNA NPs could selectively deliver siRNA to A549 cells overexpressing nucleolin via a nucleolin-mediated transmembrane process, subsequently resulting in efficient RNAi. These results indicated that the Apt-GS/siRNA NPs could facilitate nucleolin-targeted siRNA delivery and gene silencing in tumours.