A protein separation technology using the microfluidic device was developed for the more rapid and effective analysis of target protein. This microfluidic separation system was carried out using the aqueous two-phase system (ATPS) and the ionic liquid two-phase system (ILTPS) for purification method of the protein sample, and the three-flow desalting system was used for the removal of salts from the sucrose-rich sample. Partitioning of the protein sample was observed in ATPS or ILTPS with the various pHs. The microdialysis system was applied to remove small molecules, such as sucrose and salts in the microfluidic channel with the different flow rates of buffer phase. A complex purification method, which combines microdialysis and ATPS or ILTPS, was carried out for the effective purification of bacteriorhodopsin (BR) from the purple membrane of Halobacterium salinarium, which was then analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis and matrix-assisted laser desorptionionization time-of-flight. Furthermore, we were able to make a stable three-phase flow controlling the flow rate in the microfluidic channel. Our complex purification methods were successful in purifying and recovering the BR to its required value.