This paper examines the ability to measure the velocity of both physiological saline (PS) and in vitro blood in a rectangular polydimethysiloxane (PDMS) microchannel by means of the confocal micro-PIV system. The PDMS microchannel, was fabricated by conventional soft lithography, had a microchannel near to a perfect rectangular shape (300μm wide, 45μm deep) and was optically transparent, which is suitable to measure both PS and in vitro blood using the confocal system. By using this latter combination, the measurements of trace particles seeded in the flow were performed for both fluids at a constant flow rate (Re=0.021). Generally, all the velocity profiles were found to be markedly blunt in the central region mainly due to the low aspect ratio (h/w=0.15) of the rectangular microchannel. Predictions by a theoretical model for the rectangular microchannel have showed fairly good correspondence with the experimental micro-PIV results for the PS fluid. Conversely, for the in vitro blood with 20% haematocrit, small fluctuations were found on velocity profiles.