Magnetophoretic field flow fractionation (FFF) is a promising technique for bioseparation in microfluidic channels for micro-total analysis systems (μ-TAS) applications. A numerical study of FFF of magnetic microspheres of two different sizes in a micro-channel under a transverse magnetic field is studied. Due to the difference in the particle magnetophoretic mobility, particles develop different transverse velocities leading to their separation through two different outlets of the microchannel. Capture efficiency and separation index for the particles are computed at the two outlets, and their variations under different parametric conditions, viz., particle size and susceptibility, magnetic dipole strengths and positions, fluid viscosity and flow velocity, are characterized. Parametric studies show that the capture efficiency and separation index figures are high within a very narrow zone of operating conditions. Beyond this regime, the particles are either trapped on the wall before or after their designated outlets (leading to poor capture efficiency), or are getting collected at the non-designated outlet (leading to poor separation index). Therefore, it is important to determine the optimum parametric and operating condition to achieve the best performance of the FFF device for any BioMEMS application.