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Nonspecific adsorption of non-target components (especially proteins) in food matrix on sensing surfaces deteriorates accuracy and sensitivity of electrochemical sensors, which is a key challenge for food safety sensing. Therefore, electrochemical sensors with antifouling capability of high-protein food matrices are highly desired. In this paper, an efficient and simple antifouling electrochemical aptasensor for tetracycline (TC) analysis was constructed based on a self-designed Y-shaped peptide [CPPPPEK-(RSERSERSE) 2 ] consisting of cysteine as the anchoring segment, polyproline as the supporting segment, and -EK-(RSERSERSE) 2 as antifouling branches. Hydrophilic and electrically neutral amino acid sequence and Y-shaped space structure of the peptide endow the sensing surface with good antifouling performance in protein solutions and diluted milk. Under optimized experiment conditions, the proposed aptasensor can detect TC with a wide linear range (0.01–100 ng mL−1) and a limit of detection of 5.3 pg mL−1 (S/N = 3). The spiked recovery experiments confirmed high accuracy of the aptasensor in diluted milk without other pretreatments (recoveries: 90.96%–95.86%). This strategy offers a valid way to reduce matrix interference, showing a promise to be extended to other sensing systems for different targets. [Display omitted] • An innovative electrochemical aptasensor was developed for TC determination. • It consists of CPPPPEK-(RSERSERSE) 2 and specific aptamer. • The Y-shaped peptide endowed the aptasensor with excellent antifouling ability. • The proposed sensor has satisfactory selectivity, repeatability and stability. [ABSTRACT FROM AUTHOR]