The sensitive and accurate detection methods for PEDV antibody have practical significance for the prevention and treatment of PEDV. In this work, a new multiple pathways signal amplification method was proposed to construct a sensitive electrochemiluminescence (ECL) platform for the detection of PEDV antibody. Using Au NP-modified graphene nanosheet (Au-GN) as the substrate, antibody-antigen reaction as the recognition unit, rolling circle amplification (RCA) for signal enhancement, and assembled cascade Ru-DNA nanotags as signal label, the proposed platform behaved with good specificity and sensitivity. The binding system of biotin-streptavidin, RCA, and Ru(bpy)32+-doped silica nanoparticles (Ru SNPs) showed remarkable amplification efficiency, low background signal, and little nonspecific adsorption. Moreover, the proposed ECL sensor exhibited good analytical performance for PEDV antibody with a wide linear range from 0.1 pg mL-1 to 5000 pg mL-1 with a detection limit of 0.05 pg mL-1 ( S/ N = 3). The proposed strategy exhibited the advantages of excellent stability and sensitivity for determination of the PEDV antibody, which was easy to prepare and had a good application prospect.