Kanamycin (KanR) is a widely used antibiotic in human and veterinary medicine, as well as in food production and livestock breeding. However, its environmental residue and bioaccumulation in the food chain pose a great threat to human health. A real-time and sensitive aptasensor is developed for KanR detection based on a gold interdigitated electrode (IDE). A microfluidic alternating current electrothermal (ACET) effect is employed for rapid directional manipulation and enrichment of KanR molecules. As an ultrasensitive indicator, solid-liquid capacitance is adopted to reflect the tiny change on the IDE surface caused by target adsorption. The overall detection takes only 60 s from sample to result, and a wide linear detection range of 0.1 fM~1 pM, an ultra-low detection limit of 16.56 aM, and a high selectivity of 7752:1 are simultaneously achieved, with 5 times of repeated use and the shelf life of 10 days. Furthermore, the aptasensor shows excellent practicability in milk samples, with the spiked recovery rate ranging from 86.90% to 116.17%. This aptasensor with the detecting strategy provides a rapid, convenient, and cost-effective solution for real-time monitoring of KanR.