An impedance biosensor based on interdigitated electrode (IDE) arrays was designed, fabricated and tested for detection of Escherichia coli O157:H7. The device consists of two sets of gold IDE arrays embedded in a SU8-PDMS microchannel. The first set of electrodes uses positive dielectrophoresis (p-DEP) force to focus and concentrate the E. coli into the centre of the microchannel, and direct it towards the detection zone microchannel which has dimensions of a third of the first channel. The bulk fluid keeps flowing toward the outer channel into the waste outlets. The second sets of electrodes are located in the centre channel and are used for impedimetric detection of the E. coli. A combination of standard photolithography, wet etching and plasma treatment techniques were used to fabricate the biosensor. The E. coli cells in the test solution were focused into the centre of the channel when an excitation signal of 5 V_p–p at 5.6 MHz was applied across the electrode arrays. Before injecting the E. coli cells, polyclonal anti-E. coli antibodies were non-specifically immobilized on the sensing electrode array. This ensures specific detection of E. coli O157:H7 bacterial cells. As the concentrated E. coli cells (antigen) reach the sensing electrode array, they bind to the immobilized antibody sites. This antigen–antibody binding causes a change in the impedance, which is measured using an impedance analyzer. The device performance was tested by measuring the impedance, between 100 Hz and 1 MHz frequency, before and after applying p-DEP on the focusing electrode array, and after applying p-DEP on both the focusing and sensing electrodes. The result shows clearly that the use of p-DEP on the focusing IDE array significantly increased the measurement sensitivity with the lower detection limit being 3 × 10² CFU mL⁻¹. In addition, the use of p-DEP on both electrode arrays increased the measurement sensitivity by a factor of 2.9 to 4.5 times depending on the concentration.