Abstract: A fluorescent and label-free assay for the detection of kanamycin was developed based on kanamycin aptamer (Apt-DNA) and SYBR Green I (SG). Apt-DNA combines with complementary oligonucleotide chain (Pri-DNA) to form double stranded DNA (dsDNA). Although the free-state SG emits a weak fluorescence, the fluorescence intensity is significantly enhanced after attaching to dsDNA. When kanamycin was added into the solution, Apt-DNA specifically bind with kanamycin and the concentration of dsDNA is dramatically reduced, which obviously decrease the fluorescence intensity. By comparing the fluorescence intensity before and after adding kanamycin, the label-free detection of kanamycin is realized. Some advantages of this method are presented, such as simple principle, convenient operation, high sensitivity and low cost. What' more, the proposed method could be used to measure kanamycin in real samples.