In this work, a sensitive microfluidic origami electrochemical immunodevice (denoted as mu-OECI in this work) has been developed, based on polyaniline-Au nanoparticles (AuNPs) modified paper working electrode (PANI/Au-PWE) as sensor platform and different redox substrates as tracers. Au-PWE was fabricated through the growth of an AuNPs layer on the surfaces of cellulose fibers in the PWE. Through electropolymerization of aniline in an Au-PWE, the effective surface area and the sensitivity of the PANI/Au-PWE were enhanced remarkably. 3D graphene sheets (GS) with high surface-to-volume reaction and good in-plane conductivity were used as matrices to immobilize the redox probes and antibodies. Each biorecognition event yielded a distinct voltammetric peak, whose position and size reflected the identity and level of the corresponding antigen. The large amount of redox probes on the 3D GS greatly amplified the detection signals, and the good biocompatibility of PANI/Au-PWE retained good stability for the sandwich-type immunoassay. Using carcinoembryonic antigen and alpha-fetoprotein as model analytes, the simultaneous multiplex immunoassay showed linear ranges of over 4 orders of magnitude with the detection limits down to 0.5 and 0.8 pg.mL(-1), respectively. This facile biosensing mu-OECI exhibited high sensitivity and specificity, and it was applied in point-of-care testing of other cancer markers for remote regions and developing countries. (C) 2014 Elsevier Ltd. All rights reserved.