A detailed measurement of the blood flow velocity profile in microchannels in vitro is fundamental to better understand the biomechanics of microcirculation. Therefore it is very important to determine the influence of suspended blood cells on the flow behaviour with high accuracy and spatial resolution. We measured the flow of blood cells suspended in a physiological fluid within a square microchannel using a confocal particle image velocimetry (PIV) system and compared it to pure water. This emerging technology combines a conventional PIV system with a spinning confocal microscope and has the ability to obtain high-resolution images and three-dimensional (3D) optical section velocity measurements. The good agreement obtained between the measured and estimated results suggests that macroscale flow theory can be used to predict the flow behaviour of a homogeneous fluid within a 100 µm square microchannel. Our results also demonstrated the potential of the confocal system for generating 3D profiles and consequently obtaining detailed information on microscale effects in microchannels using both homogeneous and non-homogeneous fluids, such as a suspension of blood cells. Furthermore, the results obtained from our confocal micro-PIV system show the ability of this system to measure velocities up to 0.52 mm s−1 in a blood cell suspension fluid.