Practical biosensors require miniaturized device and simplified system. These requirements are fulfilled by fabricating 3-dimensional (3D) interdigitated array (IDA) electrodes and eliminating reference electrode. In this presentation, we demonstrate electrochemical immunosensors based on 2-electrode (2E) system operated by redox cycling on two working electrodes without reference electrode and counter electrode. The redox cycling between two IDA electrodes, on the ceiling as well as the bottom of the microchannel, can make faradaic signal effectively amplified. In the proposed 2E system, electrochemical potential is determined by an immobilized electron transfer mediator, poly(methylene green) (PMG) on indium-tin oxide electrode surface. PMG works as a good mediator for redox of 4-aminophenol, which is the enzymatic product for immunosensing. Harnessing such a 3D IDA immunosensor of 2E operating system, we detected human Creatine Kinase-MB, a specific biomarker of myocardial damage, down to ~ pg/mL level. Moreover, this system has the potential that multiple 3D IDA electrodes embedded in a single chip can simultaneously analyze several mixed biomarkers and can be miniaturized small enough to be portable.