In this work, we design a novel microfluidic chip to analyze and simulate the thermophoretic isolation of circulating tumor cells. For the first time, separation of circulating tumor cells from same size peripheral blood cells is examined by thermophoresis. Moreover, a discrete heat source was used to attenuate the separation efficiency, instead of a continuous heat source. Physical properties, such as thermal conductivity, gravity and hydrodynamic forces, were used in numerical design of a microfluidic chip to preferably move white blood cells toward colder walls, due to thermophoresis. To examine the separation process, or differentiated upward migration of cells between the fluid layers, the creeping flow and continuity equations are simultaneously solved along with the constituent forces by FEM modeling. Results show that upon applying a minimum temperature difference of 1 °C, white blood cells are effectively separated from tumor cells, in a 4.5-mm-long microchannel. Maintaining an oscillating/symmetrical temperature gradient in the longitudinal direction minimizes the required separation length of the channel. Moreover, for samples with relatively wide range of size distributions, thermophoresis can robustly separate the analytes, even for the same diameter analytes where the difference in buoyancy or gravity forces is infinitesimal or not present. Such small temperature difference in walls does not denature cells, the overall design is relatively cheap to apply and requires simple fabrication, and the separation is implemented label-free.