To better understand the physical mechanism of leukocyte separation via microfluidics, a level set method was employed to analyze the coupled deformation-flow of individual leukocytes in microfluidic parabolic shear blood flow. The results show that: (1) Weber number and viscosity ratio have great effects on the deformation of single leukocyte, (2) difference between the deformation and motion behavior of different subtypes of leukocytes (i.e., granulocytes, lymphocytes, monocytes) was observed, and (3) the existence of a second leukocyte significantly changes the leukocyte deformation and motion. These results shed light on the understanding of the motion and deformation of leukocytes in microchannel flow and provide a theoretical foundation for separating lymphocytes via microfluidics.