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Matrix interference resulting from the nonspecific adsorption of non-target components, particularly proteins (fouling), onto sensor surfaces poses a persistent challenge in electrochemical detection of food hazards. The development of antifouling sensor surfaces presents a viable approach to mitigate nonspecific adsorption. In this study, a novel antifouling electrochemical aptasensor, utilizing a zwitterionic polymer, was developed for the sensitive, accurate, and selective detection of tetracycline (TC) in milk. This sensor employs a poly (dopamine)-poly (sulfobetaine methacrylate) (PDA-PSBMA) antifouling copolymer, which is synthesized through an in-situ initiated copolymerization of dopamine on the sensor's surface. Subsequently, the thiol-containing aptamers were immobilized onto the PDA-PSBMA coating through a Michael addition reaction with the poly(dopamine). The resulting antifouling electrochemical aptasensor exhibited robust antifouling performance in various single protein solutions and diluted milk samples, coupled with sensitive and selective recognition of TC. The sensor demonstrated a broad linear response range of 0.1–1000.0 ng mL−1 and a low limit of detection (LOD) of 68.0 pg mL−1. The antifouling electrochemical aptasensor proved effective in assaying TC in diluted milk, with recoveries ranging from 100.0 % to 104.4 %, eliminating the need for additional pretreatments due to its exceptional resistance to nonspecific adhesion. [Display omitted] • DA self-polymerization initiated free radical polymerization of SBMA. • One-step prepared PDA-PSBMA copolymer showed excellent antifouling ability. • Thiol-containing aptamer was introduced by a Michael addition reaction with the PDA. • An antifouling electrochemical aptasensor based on PDA-PSBMA was proposed. • Sensitive TC analysis in diluted milk can be realized without other pretreatments. [ABSTRACT FROM AUTHOR]