A 3D electrochemiluminescence (ECL) origami immunodevice for highly sensitive detection of carcinoma antigen 125 (CA125) was designed based on in situ enzymatic reaction to generate H2O2 as coreactant of peroxydisulfate solution with Pd@Au nanoparticles (NPs) as catalyst for signal amplification. The novel flower-like Ag@Au hybrids modified porous paper working electrode (Ag@Au-PWE) was prepared by a two-step growth approach and served as sensor platform for antibody attachment. Glucose oxidase (GOD) and signal antibody labeled Pd@Au NPs were linked to the Ag@Au-PWE surface via sandwich immunoreactions. Integrating the dual-signal amplification strategy, the proposed method exhibited high sensitivity and specificity for the detection of CA125 with a linear range of 0.1 mU.mL(-1) to 100 U.mL(-1) with a detection limit of 0.06 mU.mL(-1). Moreover, the facile immunodevice showed excellent stability and reproducibility in real human serum assay, indicating its wide range of potential applications in point-of-care testing. (C) 2014 Elsevier Ltd. All rights reserved.