Detection of harmful biological substances in food at the Point of Use (PoU) is very important to the prevention of foodborne diseases. Sample and reagent preparation at the PoU, as a necessary step before detection, is urgently needed. Portable and field-deployable sample preparation microfluidic devices for manipulating particles and biological substances in Newtonian fluids like water have been developed. However, understanding the mechanism of particle focusing and multiplex sorting of particles with various properties in prominent non-Newtonian fluids such as milk, blood, and DNA solutions have lagged behind. Accordingly, we developed a microfluidic device to investigate the effect of inertial, magnetic and elastic forces on the focusing of magnetic (9 and 15 µm) and non-magnetic (15 µm) particles in synthetic viscoelastic fluids with various viscosities. We also demonstrated the application of our studies in Triplex Inertia-Magneto-Elastic (TIME) sorting of these particles with purity and efficiency of more than 92%. The device included a square microchannel with a side permanent magnet, expanding symmetrically downstream to a wider channel to drop the particles velocity for on-chip imaging. We investigated the effect of multiple parameters on the focusing of each particle experimentally and analytically, in order to obtain understanding of particle dynamics and the best recipe in which triplex particle separation could be achieved with high efficiency. The studied parameters included the microchannel dimension, flow rate, fluid viscoelasticity, and magnetic field strength and exposure time. This study provides the foundation for development of devices for separation of bio-substances in viscoelastic fluids, immunologically attached to microparticles. Our device has the potential to be used for on-site sample preparation along with a variety of biosensors to render biodetection possible at the PoU.