To combat serious nonspecific adsorption in complex biological media, an elegantly designed antifouling electrochemical aptasensor was fabricated for cancer biomarker-adenosine triphosphate (ATP) detection. The aptasensor utilized polydopamine-silver nanoclusters (PDA-Ag) nanoplatform as matrix. First, PDA-Ag nanoplatform with high specific surface area and numerous functional groups was formed on a glassy carbon electrode (GCE) surface via straightforward drop-coating and self-polymerization. Then, catechol & zwitterion-bifunctional poly(ethylene glycol) (b-PEG) and ATP aptamer were simultaneously self-assembled onto the PDA-Ag nanoplatform. The synthetized b-PEG could tether to diverse substrate surfaces under mild conditions utilizing catechol as the anchoring groups. Furthermore, due to the zwitterionic property, b-PEG could effectively reduce nonspecific adsorption, leading to the enhancement of the sensing selectivity and sensitivity. As a result, the detection limit of aptasensor was 0.01 pM with the linear range of 0.01 pM to 10 nM. Futher determination of ATP in human plasma also showed satisfying results. The present strategy employing bifunctional antifouling polymer enlightens a new thought to construct antifouling biosensors and holds extensive application prospects in bioanalysis and clinical diagnosis.