We report herein the first oligonucleotide-based luminescent method for the sensitive and selective detection of kanamycin in aqueous solution. The assay utilizes a square-planar luminescent platinum(II) complex and the kanamycin aptamer. The luminescence signal of the platinum(II) complex with the oligonucleotide is weak in the absence of kanamycin. Upon the addition of kanamycin, the aptamer changes from a random-coiled structure into a specific conformation containing a hairpin region. This facilitates intercalation of the platinum(II) complex into the bound aptamer, thereby resulting in an enhanced luminescence signal of the system in response to kanamycin. The assay is sensitive and selective towards kanamycin over other antibiotics. The assay could be used to detect trace levels of kanamycin in fish extracts with a comparable sensitivity to aqueous buffer solution.