A “light-up” fluorescence method was developed for the quantitative analysis of kanamycin based on DNA template synthesized silver nanoclusters(AgNCs).The silver nanoclusters with weak fluorescence were synthesized with DNA as template.A hairpin structure was formed to make the G and C bases in the aptamer sequence of DNA template close to the silver nanoclusters when the target kanamycin was identified by the aptamer sequence,which could result in the increase of fluorescence of silver clusters.Meanwhile,the change of secondary structure caused the silver nanoparticles to transform into smaller silver nanoclusters,which also enhanced the fluorescence of silver clusters.A “light-up” fluorescence method was established based on this mechanism.There was a linear relationship between fluorescence intensity ratio of silver nanoclusters and kanamycin in the concentration range of 0.075-8.0 μmol/L with a calibration curve of F/F0 =0.616 8C+0.927 6,and a detection limit of 36 nmol/L.The spiked recoveries for kanamycin in milk and honey samples ranged from 92.4% to 112%.In addition,with the increase of kanamycin concentration,the orange fluorescence of silver nanoclusters was deepened gradually,which could be used for the visual semi-quantitative detection of kanamycin.With good sensitivity and selectivity,the method could be used in the quantitative determination of trace kanamycin in complex food samples.