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 H 2 O 2 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.