Biological flows, especially blood flow, have attracted a great deal of attention from fluid engineering and hemodynamic investigation fields with advances in bio-technology. The flow of blood carries dissolved gases, nutrients, hormones, and metabolic waste through the circulatory system in the human body. In the present study, the characteristics of blood flow inside a microchannel are investigated by using a micro-particle image velocimetry (micro-PIV) and an optical image processing technique. The motion of red blood cells (RBCs) was visualized with a high-speed CCD camera. The microchannel is made of polydimethylsiloxane (PDMS) material and a slide-glass is attached to the top. The thickness of the margin cell depletion layer is calculated from an acquired raw image through the image processing method, with variations in microchannel width.