Abstract A novel signal amplification strategy for ultrasensitive multiplexed detection of cancer biomarkers using a paper-based microfluidic electrochemical immunodevice is described. Specifically, a controlled radical polymerization reaction is triggered after the capture of target molecules on the immunodevice surface. Growth of long chain polymeric materials provides numerous sites for subsequent horseradish peroxidase (HRP) coupling, which in turn significantly enhances electrochemical signal output. The signal was further amplified through the use of graphene to modify the immunodevice surface to accelerate the electron transfer. Activators generated electron transfer for atom transfer radical polymerization (AGET ATRP) was used in this study for its high efficiency in polymer grafting and better tolerance toward oxygen in air. Glycidyl methacrylate (GMA) was examined to provide excess epoxy groups for HRP coupling. In the electrochemical immunodevice, eight carbon working electrodes, as well as their conductive pads, were screen-printed on a piece of square paper, and the same Ag/AgCl reference and carbon counter electrodes were shared with another piece of square paper via stacking. Using the HRP-O-phenylenediamine-H2O2 electrochemical detection system, four cancer biomarkers: carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), cancer antigen 125 (CA125), and carbohydrate antigen 153 (CA153) were detected. A limit of detection of 0.01, 0.01, 0.05 and 0.05ngmL−1 was demonstrated, respectively. The results show that the proposed strategy offers great promises in providing a sensitive and cost-effective solution for biosensing applications.