With the increasing incidence and prevalence of antibiotic contamination in animal-derived food and drug resistance around the world, early and specific detection of antibiotic residues has garnered significant attention. Herein, for the first time, we devise a facile, label free and portable aptasensor for quantitative determination of Kanamycin (KANA) by electrochemical impedance spectroscopy (EIS), which accompanying the assembly of in-vitro selected single strand DNA (ssDNA) anti-KANA-aptamer functionalized screen printed carbon electrodes (SPCEs) used as transducer. The target detection is based on specific recognition by KANA-aptamer covalently immobilized on SPCEs surface. The surface morphology and electrochemical properties of aptasensor were characterized using Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), atomic force microscopy (AFM), cyclic voltammetry (CV) and EIS. Under optimized experimental conditions, the devised aptasensor exhibited a dynamic range 1.2-600 ngmL(-1) with linearity 1.2-75 ngmL(-1) and limit of detection (LOD) 0.11 ng mL(-1) (S/N =3) of KANA. The developed aptasensor are endowed with a good selectivity and specificity to KANA without interference from competitive analogues (streptomycin and gentamicin). For practical application, the aptasensor performance was verified in spiked milk samples and the acceptable recovery percentage of 96.88-100.5% (%RSD = 4.56, n=3) was obtained in milk samples. (C) 2017 Elsevier B.V. All rights reserved.