A novel alternating current (ac)-dielectrophoretic (DEP) microfluidic chip for continuous cell characterization and separation is presented in this paper. To generate DEP forces, two electrode-pads are embedded in a set of asymmetric orifices on the opposite sidewalls to produce the nonuniform electric fields. In the vicinity of a small orifice, the cells experience the strongest nonuniform gradient and are drawn toward it by the positive DEP forces, while the cells experiencing a negative DEP force are repelled away and move toward the large orifice. The DEP behaviors of yeast cells in suspending media with different ionic concentrations, i.e., different electrical conductivities, and over a large range of the ac electric field frequency were investigated. Furthermore, the lateral migrations of yeast cells as a function of the ac frequency were measured. The trends of measured lateral migrations of yeast cells are similar to the corresponding Clausius-Mossotti (CM) factors. In addition, by adjusting the frequency and strength of the ac electric field, the continuous separation of live and dead yeast cells as well as the yeast cells with targeted diameter and dielectric property can be easily achieved. This is the first time that the measurement of ac-DEP lateral migration of yeast cells in solutions with different electrical conductivities as a function of the applied frequency in a microfluidic chip was reported. This ac-DEP system provides a method to characterize the crossover frequency of the specific cells and manipulate the targeted cells.