Electric field-flow fractionation (EFFF) is a separation technique that couples a lateral electric field with axial Poiseuille flow to separate particles on the basis of size and/or mobility. In unidirectional EFFF, the field rapidly decreases in time due to charging of the double layer. The field strength could be increased by performing EFFF with cyclic electric fields. In cyclic electric field-flow fractionation (CEFFF), a periodic voltage, which can be either sinusoidal or square-wave, is applied in the lateral direction. In this paper, we measure the electrochemical response of CEFFF, i.e., the current-time response for a given time-dependent voltage and then utilize this electrochemical response in a transport model to predict separation. The CEFFF device studied here comprises two gold-coated glass plates separated by a spacer. The transient current profiles are measured for a step change and cyclic square-shaped voltage. The current profile is compared with the equivalent circuit model, and is fitted to a sum of two decaying exponentials. The dependence of the electrochemical response on voltage, frequency, channel thickness, and salt concentration is studied. Next, the electrochemical data are utilized in the convection-diffusion equation to develop a model for separation by CEFFF. The equations are solved by using a combination of analytical and numerical techniques to determine the mean velocity and the dispersion coefficient of molecules, and to determine the effect of various parameters on the separation efficiency of the EFFF device. Also, the model predictions are compared with experimental data available in the literature.