In this study, we investigated the particle separation phenomenon in a microchannel with a T-shaped cross-section, a unique design detailed in our previous study. Utilizing a co-flow system within this T-shaped microchannel, we examined two types of flow configuration: one where a Newtonian fluid served as the inner fluid and a viscoelastic fluid as the outer fluid (Newtonian/viscoelastic), and another where both the inner and outer fluids were Newtonian fluids (Newtonian/Newtonian). We introduced a mixture of three differently sized particles into the microchannel through the outer fluid and observed that the co-flow of Newtonian/viscoelastic fluids effectively separated particles based on their size compared with Newtonian/Newtonian fluids. In this context, we evaluated and compared the particle separation efficiency, recovery rate, and enrichment factor across both co-flow configurations. The Newtonian/viscoelastic co-flow system demonstrated a superior efficiency and recovery ratio when compared with the Newtonian/Newtonian system. Additionally, we assessed the influence of the flow rate ratio between the inner and outer fluids on particle separation within each co-flow system. Our results indicated that increasing the flow rate ratio enhanced the separation efficiency, particularly in the Newtonian/viscoelastic co-flow configuration. Consequently, this study substantiates the potential of utilizing a Newtonian/viscoelastic co-flow system in a T-shaped straight microchannel for the simultaneous separation of three differently sized particles.