Particle flow separation is a useful technique in lab-on-a-chip applications to selectively transport dispersed phases to a desired branch in microfluidic devices. The present study aims to demonstrate both nano- and microparticle flow separation using microscale thermophoresis at a Y-shaped branch in microfluidic channels. Microscale thermophoresis is the transport of tiny particles induced by a temperature gradient in fluids where the temperature variation is localized in the region of micrometer order. Localized temperature increases near the branch are achieved using the Joule heat from a thin-film micro electrode embedded in the bottom wall of the microfluidic channel. The inlet flow of the particle dispersion is divided into two outlet flows which are controlled to possess the same flow rates at the symmetric branches. The particle flow into one of the outlets is blocked by microscale thermophoresis since the particles are repelled from the hot region in the experimental conditions used here. As a result, only the solvent at one of outlets and the residual particle dispersion at the other outlet are obtained, i.e., the separation of particles flows is achieved. A simple model to explain the dynamic behavior of the nanoparticle distribution near the electrode is proposed, and a qualitative agreement with the experimental results is obtained. The proposed method can be easily combined with standard microfluidic devices and is expected to facilitate the development of novel particle separation and filtration technologies.