Microfluidic image cytometry is developed and validated both theoretically and experimentally, which is a method for the simultaneous measurement of the number and sizes of particles flowing through a microchannel using image sequence analysis and micro particle image velocimetry technique. Theoretical considerations on image formation in this method predict the image profile of a known particle and suggest that corrections are required in particle size measurement in order to cancel the effects of both diffraction and out-of-focus location. A dilution series of 2 µm polystyrene particle suspensions were measured and compared with the results obtained by conventional Bürker–Türk hemocytometry for validation of the particle counting. For the particle diameter measurements, the diameters of 2, 5, 10 and 20 µm particles were measured and compared with the official values of the manufacturer. The results of the number and sizes of the particles measured by the proposed method agreed well with the reference values. We hope that the proposed method will be applicable to the quantitative study of platelet aggregation in blood flow and become a powerful diagnostic tool in the future.