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In antibiotics, β-lactam is one kind of major concern acknowledged as an unavoidable contaminant in milk. Thus, a facile and sensitive method is essential for rapid β-lactam antibiotics detection. In our work, a specific electrochemical receptor sensor based on the graphene/thionine (GO/TH) composite was established. The mechanism of the electrochemical receptor sensor was a direct competitive inhibition of the binding of horseradish peroxidase-labeled ampicillin (HRP-AMP) to the mutant BlaR-CTD protein by free β-lactam antibiotics. Then, horseradish peroxidase (HRP) catalyzed the hydrolysis of the substrate hydrogen peroxide (H(2)O(2)), which produced an electrochemical signal. Under optimal experimental conditions, this method could quantitatively detect cefquinome from 0.1 to 8 μg L(−1) and with the limit of detection (LOD) of 0.16 μg L(−1), much lower than the maximum residue limit (MRL) of 5 μg L(−1) set by the European Union. In addition, the LOD of spiked milk samples with cefalexin, cefquinoxime, cefotafur, penicillin G and ampicillin were 14.88 μg L(−1), 2.46 μg L(−1), 17.16 μg L(−1), 0.06 μg L(−1), 0.21 μg L(−1) and the limits of quantitation (LOQ) were 36.09 μg L(−1), 5.40 μg L(−1), 41.45 μg L(−1), 0.13 μg L(−1), 0.42 μg L(−1), respectively. The sensor showed a favorable recovery of 84.89–102.44%. Moreover, the electrochemical receptor sensor was successfully applied to assay β-lactam antibiotics in milk, which showed good correlation with the results obtained from liquid chromatography-tandem mass spectrometry (LC-MS/MS).