Selective and sensitive determination of trace kanamycin in complex food samples is of great importance for food safety because of its high toxicity. Here, we report a sensitive and autofluorescence-free persistent luminescence (PL) aptasensor for selective, sensitive, and autofluorescence-free determination of kanamycin in food samples. The aptamer for kanamycin was first conjugated onto the surface of magnetic nanoparticles Fe3O4 to serve as the recognition unit as well as the separation element, while the PL nanoparticles ZnGa2O4:Cr (PLNPs) were functionalized with the aptamer complementary DNA (cDNA) as the PL signal. The PL aptasensor consisted of the aptamer-conjugated MNPs (MNPs-apt) and cDNA-functionalized PLNPs (PLNPs-cDNA) and combined the merits of the long-lasting luminescence of PLNPs, the magnetic separation ability of MNPs as well as the selectivity of the aptamer, offering a promising approach for autofluorescence-free determination of kanamycin in food samples. The proposed aptasensor showed excellent linearity in the range from 1 pg mL-1 to 5 ng mL-1 with a limit of detection of 0.32 pg mL-1. The precision for 11 replicate determinations of 100 pg mL-1 kanamycin was 3.1% (relative standard deviation). The developed aptasensor was applied for the determination of kanamycin in milk and honey samples with the recoveries of 95.4-106.3%. The proposed aptasensor is easily extendable to other analytes by simply replacing the aptamer, showing great potential as a universal aptasensor platform for selective, sensitive, and autofluorescence-free detection of hazardous analytes in food samples.