Abstract: Small interfering RNA (siRNA) often acts as an important therapeutic agent in gene therapy due to its ability of reducing gene expression. However, the low cell uptake rate of siRNA limits its efficacy in cancer treatment. Here, we report a biocompatible nanomotors-hydrogel system to obtain high cell uptake rate for targeted cancer therapy. Nanomotors (NM) loaded with siRNA were prepared employing polyethyleneimine (PEI) and sodium polystyrene sulfonate (PSS) via layer-by-layer self-assembly technology. For the sake of intratumoral administration and slow release, the nanomotors were loaded in Schiff-base hydrogel to build NM-hydrogel system. Taking advantage of the tumor microenvironment featured with an acidic pH and a high content of hydrogen peroxide, the hydrogel releases nanomotors in response to slightly acidic tumor matrix, and the released nanomotors can autonomously move through catalytic decomposition of hydrogen peroxide, the speed of which can be maintained at 1.78 µm/s at 1wt%H₂O₂ (22.25 body lengths per second). The autonomous motion of nanomotors and specific endocytosis which is mediated by folic acid (FA) modified on the nanomotors gives the NM-hydrogel system a high cell uptake rate of 63.8%. The proton sponge effect caused by PEI and autonomous motion promote the deep penetration and long-time retention of namomotors in tumor cells, which facilitates the anticancer effect of the NM-hydrogel system. The results show that the anticancer activity of the system is 74.8% at 72 h. Meanwhile, the NM-hydrogel system has good biocompatibility and biodegradability, which lays the foundation for its future application in gene therapy in vivo.