Abstract A label-free detection method of kanamycin using aptamer-based cantilever array sensor was developed. The cantilever array was composed of sensing cantilevers and reference cantilevers. This configuration allowed direct detection of individual cantilever deflections and subsequent determination of differential deflection of sensing/reference cantilever pair. The sensing cantilevers were functionalized with kanamycin aptamer, which was used as receptor molecules while the reference cantilevers were modified with 6-mercapto-1-hexanol (MCH) to eliminate the influence of environmental disturbances. The kanamycin–aptamer interaction induced a change in cantilever surface stress, which caused a differential deflection between the sensing and reference cantilever pair. The surface stress change was linear with kanamycin concentration over the range of 100μM–10mM with a correlation coefficient of 0.995. A detection limit of 50μM was obtained, at a signal-to-noise ratio of 3. The sensor also showed good selectivity against other antibiotics such as neomycin, ribostamycin and chloramphenicol. The facile method for kanamycin detection may have great potential for investigating more other molecules.