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A highly active G4 DNAzyme-peptide complex obtained by covalent assembly of cationic peptide on G4 DNAzyme was used to construct a novel electrochemical aptasensor. The effect of complex on the performance of electrochemical aptasensor was investigated and the proposed aptasensor was used to detect kanamycin (KANA) in milk. The experimental results indicated that G4 DNAzyme-peptide could significantly amplify the electrochemical signal and greatly improve the detection sensitivity of aptasensor. Under the optimal concentration of capture probe (CP, 2.0 µmol/L), the change of current intensity showed a good linear relationship with the logarithm of the target concentration in the range of 0.06 pmol/L~20 nmol/L, and the detection limit was 0.02 pmol/L, which was superior to other KANA detection methods. The aptasensor showed better selectivity and lower detection limit and the recoveries obtained in milk sample were from 97.1% to 105.5% and the relative standard deviation were from 3.60% to 5.74%. The experimental results were consistent with ELISA method, indicating the proposed aptasensor could achieve high sensitivity for the detection of KANA in milk. [ABSTRACT FROM AUTHOR]