An enzyme-free electrochemical immunoassay is introduced into microfluidic paper-based analytical devices for the first time using novel three dimensional flower-like Au nanoparticles modified paper working electrode (FLAuNPs-PWE) as sensor platform and gold (Au)-silver (Ag) bimetallic nanoparticles (Au-Ag BMNPs) as tracer. FLAuNPs with large surface area serve as an effective matrix for primary antibodies. Au-Ag BMNPs with good biological activity, such as mimicking natural peroxidases, are used for binding of signal antibodies (Ab(2)). Then the Ab2 labeled BMNPs is linked to the FLAuNPs-PWE via sandwich immunoreactions. Enhanced sensitivity is achieved by efficient catalysis of the Au-Ag BMNPs toward hydrogen peroxide. Under the optimized experimental conditions, the proposed paper- based electrochemical immunosensor exhibits excellent analytical performance for enzyme- free detection of carcinoembryonic antigen, ranging from 0.001- 50 ng mL(1) with a low detection limit of 0.3 pg mL(1). The immunosensor shows good precision, acceptable stability and reproducibility. The proposed method provides a new promising platform for the design of the highly sensitive detection method, showing great promise for clinical application. (C) 2014 Elsevier Ltd. All rights reserved.