A new transducer for biosensor applications has been developed based on a three-dimensional interdigitated electrode array (IDEA) with electrode digits separated by an insulating barrier. Binding of molecules to a chemically modified surface of the transducer induces important changes in conductivity between the electrodes. Three-dimensional sensor shows considerable improvement compared with a standard planar IDEA design. The potential of the developed device as a sensor transducer to detect immunochemical and enzymatic reactions, as well as DNA hybridization events is demonstrated. The immunosensor allows direct detection of the antibiotic sulfapyridine and shows the IC₅₀ parameter value of 5.6μgL⁻¹ in a buffer. Immunochemical determination occurs under competitive configurations and without the use of any label. Each modified sensor is of a single use. Nevertheless, biochemical reagents can be easily cleaned off the sensor surface for its reuse. Layer-by-layer method of used to deposit polyethyleneimine and glucose oxidase showed that the sensor is also highly effective for detecting single and multilayered molecular assemblies.