The autonomous motion of chemically-powered synthetic nanomotors has attracted a significant recent interest owing to their great promise for diverse potential applications. Recent reviews highlighting the factors influencing the nanomotor speed, including the motor and fuel compositions, along with new nanomotor capabilities and prospects, have been reported elsewhere.1-3 However, such autonomously propelling nanoscale materials have not been exploited for biosensing applications. This protocol describes the preparation of synthetic nanomotors and a DNA modified chip for the purpose of transducing nucleic acid hybridization events into motion. The new motion-based transduction approach relies on measuring changes in the speed of unmodified catalytic nanomotors, induced by the dissolution of silver nanoparticle tags, captured in a sandwich nucleic acid assay in the hydrogen peroxide fuel. The presented strategy offers sensitive, easily measured distance readouts down to 40 amol DNA as well as the ability to directly detect raw Escherichia coli rRNA without isolation or purification steps. Unlike common optical or electrochemical DNA hybridization assays, the protocol described requires no particle enlargement and therefore it is not susceptible to non-specific Ag precipitation. The assay can also be measured using portable, simple and low-cost microscopic readers, making the new method affordable and attractive for low-resource settings.