Three-dimensional biochemical sensors are developed that can be used for chemical and biological detection in aqueous solutions and suspensions. The sensors are fabricated using a standard polycrystalline silicon process, PolyMUMPs, and can detect chemicals and biomarkers in low concentrations in near real time. The sensors made of a stack of electrodes allowing the solution to occupy the space between the layers of electrodes and have a larger interface with the electrodes. The sensors use electrochemistry impedance spectroscopy (EIS) for detection and therefore increasing the solution-electrode interface improves the sensitivity of the sensor. To demonstrate the applicability of the proposed sensor design, experimental measurements are used to characterize and compare the 3D sensors with conventional 2D interdigitated sensors. Diethylhexyl phthalate (DEHP) solution is used as the target chemical, and the 2D and 3D biochemical sensors are exposed to different concentrations of DEHP solution. An LCR meter is used to sweep the frequency and determine the impedance of the sensor-solution combination. The test results show that the three-dimensional sensors have higher sensitivity than 2D interdigitated ones verifying the advantage of the new sensor design over existing conventional sensors. The proposed sensors can also be used for detection of biological markers such as cells, proteins and enzymes in aqueous solutions.