We describe the development, fabrication and testing of a microfluidic chamber for dielectrophoretic field-flow separation of biological cells based on their electrical properties. The chamber was constructed from a single Pyrex wafer with interdigitated Au electrodes, a spacer, and a top cover glass, making the events in the chamber observable under most optical microscopes. The dimensions were optimized based on numerical computations of the electric field, its gradient and the fluid-flow velocity profile. The electrodes were fabricated using photolithography. A double-sided self-adhesive tape of 100 μm thickness was used as a spacer, with an opening of 80 mm length and 20 mm width cut in its middle to form a channel of 100 μm height, and with water-resistant acrylic glue of the tape holding the glass plates together and providing a tight seal. The glue loses its adhesive properties above 70 °C, allowing for easy disassembly of the chamber in hot water and its thorough cleaning. A 1:1 mixture of normal and 50 °C -heat-treated CHO cells was used to test the chamber. A 93% efficiency of separation was obtained, confirming the usefulness of the chamber in separating cells with sufficient differences in electrical properties of their membranes.