A microfluidic device was fabricated via UV lithography technique to separate non-magnetic fluoresbrite carboxy microspheres (approximately 4.5 microm) in the pH 7 ferrofluids made of magnetite nanoparticles (approximately 10 nm). A mixture of microspheres and ferrofluid was injected to a lithographically developed Y shape microfluidic device, and then by applying the external magnet fields (0.45 T), the microspheres were clearly separated into different channels because of the magnetic force acting on those non-magnetic particles. During this study, various pumping speeds and particle concentrations associated with the various distances between the magnet and the microfluidic device were investigated for an efficient separation. This study may be useful for the separation of biological particles, which are very sensitive to pH value of the solutions.