The invention discloses aptamer-based rapid kanamycin detection test paper as well as a preparation method and application thereof, and belongs to the field of analytical chemistry as well as safety detection on medicine, environment and food. The aptamer-based rapid kanamycin detection test paper comprises a sample pad, a colloidal gold pad, a nitrocellulose membrane, a water absorbing pad, a PVC bottom plate, a detection line and a quality control line. By the preparation method, by virtue of the characteristics that gold nanoparticles and silver nanoparticles are easy to prepare and modify and can dually amplify a signal as well as the conformational change of an aptamer, the strength change of an optical signal of the detection line is achieved, and thus detection on kanamycin is achieved; by using a test paper method for detecting the kanamycin, simple, convenient and fast detection is achieved, detection can be performed anytime and anywhere, a result is visualized, the detection time is short, an experimental result can be obtained within 10min by just inserting one end of the sample pad of the test paper into detection liquid, and the detection efficiency can be greatly improved; through a reading of a colloidal gold quantometer, quantitative analysis can be achieved; therefore, the aptamer-based rapid kanamycin detection test paper is of great significance in detection on kanamycin residue in animal-derived food.