This article demonstrated a chip device with alternating current (AC) dielectrophoresis (DEP) for separation of non-biological micro-particle and bacteria mixtures. The DEP separation was achieved by a pair of metal electrodes with the shape of radal-interdigital to generate a localized non-uniform AC electric field. The electric field and DEP force were firstly investigated by finite element methods (FEM). The mixed microparticles such as different scaled polystyrene (PS) beads, PS beads with inorganic micro-particles (e.g., ZnO and silica beads) and non-bioparticles with bacterial Staphylococcus aureus (S. aureus) were successfully separated at DEP-on-a-chip by an AC electric field of 20 kHz, 10 kHz and 1 MHz, respectively. The results indicated that DEP trapping can be considered as a potential candidate method for investigating the separation of biological mixtures, and may well prove to have a great impact on in situ monitoring of environmental and/or biological samples by DEP-on-a-chip.