Fluidic conditions for the separation of phases were surveyed in a microfluidic aqueous two-phase extraction system. The infusion ratio between polyethylene glycol (PEG) and dextran solution defines the concentrations of each polymer in micro-channel, which determine the phase-separation. The appropriate ratio between PEG (M.W. 8000, 10%, w/v) and dextran T500 (M.W. 500000, 5%, w/v) in order to perform the separation of phases of both polymers was observed as changing the mixed ratio of both polymers. Based on the fluidic conditions, stable two-phase solutions were obtained within 4% to 8% and 3% to 1% of PEG and dextran, respectively. In addition, the characteristics of the two-phase were discussed. The separation technique studied in the paper can be applied for the implementation of a lab-on-a chip which can detect various biological entities such cells, bacterium, and virus in an integrated manner using built in a biosensor inside the chip.