A brief and universal ultralow fouling sensing platform capable of assaying targets in complex biofluids was developed based on designed antifouling peptides that could form a loop-closed structure with enhanced stability. The newly designed peptide with thiol groups in its two terminals self-assembled onto a gold nanoparticle (AuNP)-modified electrode surface to form a stable loop structure, which displayed excellent antifouling performance, outstanding stability under enzymatic hydrolysis, and satisfactory long-term antifouling capability in complex biofluids (clinical human serum). The antifouling and highly sensitive electrochemical aptasensor was constructed via one-step co-immobilization of the designed peptides and aptamers onto the electrode surface modified with electrodeposited poly(3,4-ethylenedioxythiophene) (PEDOT) and AuNPs. The developed peptide-based aptasensor exhibited a decent response for the analysis of the cancer antigen 125 (CA125), with a relatively wide linear range (0.1-1000 U mL-1) and a low limit of detection (0.027 U mL-1), and was capable of detecting CA125 in clinical serum samples with acceptable accuracy. This antifouling strategy-based self-assembled peptide with a loop-closed structure provided a potential path for the development of various low-fouling biosensors for application in complex biological fluids.