A micro flow-channel with bottom-microgrooves has been manufactured by photolithography technique for cell sorting. The movement of each cell passing over microgrooves has been analyzed in relation to cell deformation and alignment in vitro. The flow path (height 0.05 mm × width 1 mm × length 25 mm) between the two transparent PDMS disks has rectangular microgrooves (4.5 μm deep, 0.2 mm long) on the bottom. Variations are made in groove widths (0.03 mm, 0.04 mm, and 0.05 mm). The angle between the flow direction and the longitudinal axis of the groove is 45 degrees. Myoblasts (C2C12: mouse myoblast line) were used in the flow test. The main flow velocity of the medium (0.02 mm/s < vx < 0.23 mm/s) was controlled by the pressure difference between the inlet and the outlet. The shape of each flowing cell was tracked in a movie recorded by a camera attached to the eyepiece of the microscope. Experimental results show that the movement perpendicular to the main flow direction in the micro-groove can distinguish cells in relation to smaller deformations and larger alignment changes.