An electrodeless dielectrophoretic (EDEP) chip was designed and applied to separate the micro-particles in different sizes, and the ability of bio-separation of real samples also be examined. The dielectrophoretic (DEP) force can be principally created and controlled by provide a non-uniform electric field that is geometrically constricted by the insulator-patterned chip in combination with an alternative current (AC) electric field at frequency 10 kHz and 500 Vp-p set on the two sides of channel inlet. The EDEP chip was used to separate the E. coli and red blood cells (RBC), which from human whole blood sample, via well control of DEP force. Our results showed the bacteria and RBC can be separated into the higher and lower electric field regions of the EDEP chip in few seconds, respectively. A rapid, useful diagnosis tool, based on the EDEP method could be applied and used in the various fields of the bio-industry technology, the detection and the identification of clinical infections.