We introduce three-dimensional (3D) interdigitated electrode array (IDA), which is a pair of IDAs on the ceiling and bottom in a microfluidic channel. The IDAs comprise of Indium tin oxide (ITO) microband electrodes that are alternatively biased. ITO based IDA can be easily and precisely patterned using conventional etching process to be integrated in a single microchannel in which very small volume of liquid sample is enough to fill. Correspondingly requiring extremely small amount of sample, the proposed strategy offers great opportunity for multiplex electrochemical biosensors with high sensitivity. ITO as an electrode material can make 3D IDA more attractive because the limit of detection goes down owing to substantially low background current. Additionally, this serves as a transparent electrode that allows simultaneous optical detection and thus widens the range of options for electrochemical biosensing chip. There are two key issues toward practical analysis. One is reference electrode that needs to lie as closely as possible to working electrodes to minimize iR drop for precise control of electrochemical potential. It is problematic because reference electrode can be hardly miniaturized to be put inside the microchannel with negligible iR drop due to narrow microchannel, i.e. high resistance. The other is functionalization of ITO surface for efficient plan of glass surface as well as ITO microbands. ITO is chemically inert so that few organic molecules can be anchored and any functional layer on it is readily detached to go away. In this presentation, we introduce two-electrode (2E) system that has appropriate mediator layer on ITO IDA. 2E system consists of only two complementary IDA demanding no reference electrodes while electrochemical potential can be retained owing to the mediators lying on the ITO electrodes. And we suggest a more reliable and versatile way of electrografting to firmly pin various mediators onto ITO surface for sensitive electrochemical immunosensing on a chip.