Ribonuclease H (RNase H), an intracellular ribonuclease, plays a crucial role in cellular processes and especially relates to many disease processes. Here, we report a novel signal amplification strategy based on an RNase H-powered DNA walking machine for specific and sensitive RNase H activity detection. The DNA walking machine is composed of a small quantity of DNA walker strands and abundant FAM-labeled DNA-RNA chimeric strands on a single gold nanoparticle (AuNP). RNase H can specifically degrade the RNA fragment in a DNA-RNA hybrid duplex and trigger the autonomous movement of a DNA walker strand on the AuNP surface. During this process, each step of the walking can release the FAM-labeled RNA from the surface of the AuNP, realizing the signal amplification for RNase H sensing. This method has been successfully utilized for RNase H activity detection in a complex system and applied for screening of related inhibitors. Therefore, our RNase H-powered DNA walking machine gives a novel platform for RNase H activity detection and RNase H-associated drug discovery.