We present a microfluidic device for in-flow dielectric characterization of single biological cells. The dielectric spectrum is obtained by measuring the multiple-frequency dielectrophoresis (DEP) response of individual cells as they travel over an array of sensing and actuating electrodes. The DEP induced translation of each cell is detected by measuring the differential impedance of the array using a microwave interferometer, which is capable of sub-attofarad sensitivity, and is coupled to the sensing electrodes. The DEP response of a cell at multiple frequencies in the beta-dispersion region is chosen to discern particular cell dielectric properties as it travels along the array—such as cytoplasm conductivity and membrane capacitance. The Clausius–Mossotti factor of the cell is determined from the measured response signal in conjunction with numerical simulation of its trajectory. The approach is validated through measuring polystyrene microspheres. The DEP response of Chinese hamster ovary cells using two simultaneous frequencies is demonstrated.